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Zhao Z, Feng X, Chen B, Wu Y, Wang X, Tang Z, Huang M, Guo X. CDCA genes as prognostic and therapeutic targets in Colon adenocarcinoma. Hereditas 2025; 162:19. [PMID: 39924497 PMCID: PMC11809055 DOI: 10.1186/s41065-025-00368-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2024] [Accepted: 01/13/2025] [Indexed: 02/11/2025] Open
Abstract
OBJECTIVES The study investigates the role of Cell Division Cycle Associated (CDCA) genes in colorectal cancer (COAD) by analyzing their differential expression, epigenetic alterations, prognostic significance, and functional associations. METHODOLOGY This study employed a detailed in silico and in vitro experiments-based methodology. RESULTS RT-qPCR assays reveal significantly elevated mRNA levels of CDCA2, CDCA3, CDCA4, CDCA5, CDCA7, and CDCA8 genes in COAD cell lines compared to controls. Bisulfite sequencing indicates reduced promoter methylation of CDCA gene promoters in COAD cell lines, suggesting an epigenetic regulatory mechanism. Analysis of large TCGA datasets confirms increased CDCA gene expression in COAD tissues. Survival analysis using cSurvival database demonstrates negative correlations between CDCA gene expression and patient overall survival. Additionally, Lasso regression-based models of CDCA genes predict survival outcomes in COAD patients. Investigating immune modulation, CDCA gene expression inversely correlates with immune cell infiltration and immune modulators. miRNA-mRNA network analysis identifies regulatory miRNAs targeting CDCA genes, validated by RT-qPCR showing up-regulation of has-mir-10a-5p and has-mir-20a-5p in COAD cell lines and tissues. Drug sensitivity analysis suggests resistance to specific drugs in COAD patients with elevated CDCA gene expression. Furthermore, CDCA gene expression correlates with crucial functional states in COAD, including "angiogenesis, apoptosis, differentiation, hypoxia, inflammation, and metastasis." Additional in vitro experiments revealed that CDCA2 and CDCA3 knockdown in SW480 and SW629 cells significantly reduced cell proliferation and colony formation while enhancing cell migration. CONCLUSION Overall, the study elucidates the multifaceted role of CDCA genes in COAD progression, providing insights into potential diagnostic, prognostic, and therapeutic implications.
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Affiliation(s)
- Zongquan Zhao
- Department of General Practice, Pingjiang New Town Community Health Service Center Sujin Street Gusu District, Suzho, 215000, Jiangsu, China
| | - Xinwei Feng
- Department of Digestive Internal Medicine, Shanghai Changzheng Hospital, Shanghai, 200003, China
| | - Bo Chen
- Department of Oncology, Chengdu First People's Hospital, Chengdu Sichuan, 610041, China
| | - Yihong Wu
- Department of General Practice, Runda Community Health Service Center, Wumenqiao Street, Gusu District, Suzhou, 215000, Jiangsu, China
| | - Xiaohong Wang
- Department of General Practice, Pingjiang New Town Community Health Service Center Sujin Street Gusu District, Suzho, 215000, Jiangsu, China
| | - Zhenyuan Tang
- Department of General Practice, Community Health Management Center of Suzhou Municipal Hospital, Suzhou, 215000, Jiangsu, China
| | - Min Huang
- Department of General Practice, Suzhou Municipal Hospital, Suzhou, 215000, Jiangsu, China
| | - Xiaohua Guo
- Department of Digestive Surgery, Xi'an Jiaotong University School of Medicine Affiliated Honghui Hospital, Xi'an, Shaanxi, 700054, China.
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Rivi V, Batabyal A, Benatti C, Tascedda F, Blom JMC, Lukowiak K. Quercetin, the new stress buster: Investigating the transcriptional and behavioral effects of this flavonoid on multiple stressors using Lymnaea stagnalis. Comp Biochem Physiol C Toxicol Pharmacol 2025; 287:110053. [PMID: 39442780 DOI: 10.1016/j.cbpc.2024.110053] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/05/2024] [Revised: 10/07/2024] [Accepted: 10/17/2024] [Indexed: 10/25/2024]
Abstract
Growing evidence suggests that a flavonoid-rich diet can prevent or reverse the effects of stressors, although the underlying mechanisms remain poorly understood. One common and abundant flavonoid found in numerous foods is quercetin. This study utilizes the pond snail Lymnaea stagnalis, a valid model organism for learning and memory, and a simple but robust learning paradigm-operant conditioning of aerial respiration-to explore the behavioral and transcriptional effects of different stressors on snails' cognitive functions and to investigate whether quercetin exposure can prevent stress effects on learning and memory formation. Our findings demonstrate that three different stressors-severe food deprivation, lipopolysaccharide injection (an inflammatory challenge), and fluoride exposure (a neurotoxic agent)-block memory formation for operant conditioning and affect the expression levels of key targets related to stress response, energy balance, and immune response in the snails' central ring ganglia. Remarkably, exposing snails to quercetin for 1 h before stress presentation prevents these effects at both the behavioral and transcriptional levels, demonstrating the potent stress-preventive properties of quercetin. Despite the evolutionary distance from humans, L. stagnalis has proven to be a valuable model for studying conserved mechanisms by which bioactive compounds like quercetin mitigate the adverse effects of various stressors on cognitive functions across species. Moreover, these findings offer insights into quercetin's potential for mitigating stress-induced physiological and cognitive impairments.
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Affiliation(s)
- Veronica Rivi
- Dept. of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy.
| | - Anuradha Batabyal
- Department of Physiology and Pharmacology, Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Alberta, Canada; Department of Physical and Natural Sciences, FLAME University, Pune, India
| | - Cristina Benatti
- Dept. of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy; Centre of Neuroscience and Neurotechnology, University of Modena and Reggio Emilia, Modena, Italy
| | - Fabio Tascedda
- Centre of Neuroscience and Neurotechnology, University of Modena and Reggio Emilia, Modena, Italy; CIB, Consorzio Interuniversitario Biotecnologie, Trieste, Italy; Dept. of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
| | - Johanna Maria Catharina Blom
- Dept. of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy; Dept. of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
| | - Ken Lukowiak
- Department of Physiology and Pharmacology, Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Alberta, Canada
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Ni L, Li P, Zou Q, Li F, Chen Y, Chen H, Lai J, Du J, Liu Y. Natural infection of hybrid sturgeon ( Acipenser baerii♀× Acipenser schrenckii♂) with Nocardia seriolae and white sturgeon iridovirus: pathological and transcriptomic analyses. Front Immunol 2024; 15:1488159. [PMID: 39650642 PMCID: PMC11621106 DOI: 10.3389/fimmu.2024.1488159] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2024] [Accepted: 11/01/2024] [Indexed: 12/11/2024] Open
Abstract
Introduction In August 2023, hybrid sturgeons (Acipenser baerii♀×Acipenser schrenckii♂) cultured in Sichuan, China, showed infectious disease symptoms, including ulcers, liver and spleen nodules, and high mortality rates. Methods Pathogenic bacteria were isolated from the liver of diseased sturgeons and analyzed for their phenotypic and molecular traits. Furthermore, iridovirus-specific TaqMan real-time PCR (RT-PCR) analyses were conducted. The histopathological characteristics were analyzed using paraffin sectioning and transmission electron microscopy (TEM). Transcriptome sequencing was performed to elucidate the impact of pathogen exposure and immune response profiles in infected sturgeon. Results Pathogenic bacteria isolation and phylogenetic analyses of the 16S rRNA gene confirmed that the isolated bacteria clustered within the Nocardia seriolae group. The TaqMan RT-PCR assay was performed to detect the presence of white sturgeon iridovirus (WSIV), indicating a weakly positive signal. Histopathological examination revealed severe damage to various tissues, and a notable presence of bacteria was observed through acid-fast staining. Transmission electron microscopy analysis showed the presence of abundant bacteria and virus particles, indicating cellular invasion and subsequent damage. In summary, the disease in hybrid sturgeons was diagnosed as infection of N. seriolae and WSIV. To investigate the immune response of hybrid sturgeons to this infection, spleen transcriptomes were analyzed. Numerous immune-related genes and pathways, including the "Toll-like receptor", "B-cell receptor", and "T-cell receptor" signaling pathways, were altered in response to pathogenic threats. Significantly downregulated of key components of TCR and BCR signaling pathways, such as ZAP70, BTK, and CD79A, suggested a temporary inhibition of these pathways critical for cellular immunity post-infection. Gene set enrichment analysis revealed significant suppression of the apoptosis signaling pathway and activation of autophagy and mitophagy signaling pathways following infection. Specifically, in the death receptor-mediated apoptosis signaling pathway, downregulation of TNFα, TRAIL, CASP6, and CASP8 was observed, while several genes in the autophagy and mitophagy pathways showed upregulated expression post-infection. Discussion We report the initial occurrence of N. seriolae infection in cultured sturgeons. These findings could provide a theoretical basis for diagnosing and preventing this disease, as well as enhance the understanding of host-pathogen interactions in fish.
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Affiliation(s)
- Luyun Ni
- Fisheries Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu, Sichuan, China
| | - Pengcheng Li
- Fisheries Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu, Sichuan, China
| | - Qiaolin Zou
- Fisheries Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu, Sichuan, China
| | - Feiyang Li
- Fisheries Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu, Sichuan, China
| | - Yeyu Chen
- Fisheries Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu, Sichuan, China
| | - Haoting Chen
- Guangzhou Double Helix Gene Technology Co., Ltd., Guangzhou, Guangdong, China
| | - Jiansheng Lai
- Fisheries Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu, Sichuan, China
| | - Jun Du
- Fisheries Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu, Sichuan, China
| | - Ya Liu
- Fisheries Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu, Sichuan, China
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Qiu Y, Bai Y, Wang W, Wang Q, Chen S, Zhang J. Reference Gene Selection for RT-qPCR Normalization in Toxoplasma gondii Exposed to Broxaldine. Int J Mol Sci 2024; 25:11403. [PMID: 39518956 PMCID: PMC11546418 DOI: 10.3390/ijms252111403] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2024] [Revised: 10/18/2024] [Accepted: 10/21/2024] [Indexed: 11/16/2024] Open
Abstract
Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is widely used to accurately assess target gene expression. Evaluating gene expression requires the selection of appropriate reference genes. To identify reliable reference genes for Toxoplasma gondii (T. gondii) under varying concentrations of broxaldine (BRO), we employed the ΔCt method, BestKeeper, NormFinder, GeNorm, and the comprehensive web-based platform RefFinder to assess the expression stability of ten candidate reference genes in T. gondii. Herein, our findings reveal that the stability of these candidate reference genes is influenced by different experimental conditions. Under normal conditions, the most stable genes were TGME49_205470 and TGME49_226020. However, the most stable genes differed when BRO concentrations were at 1, 2, and 4 μg/mL. Across all samples, TGME49_247220 and TGME49_235930 were identified as the most stable reference genes. Moreover, we also confirmed the stability of TGME49_247220 and TGME49_235930 as reference genes through RT-qPCR assays. The present study provides a foundation for applying the RT-qPCR method to investigate target gene expression following BRO treatment in T. gondii.
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Affiliation(s)
- Yanhua Qiu
- Key Laboratory of New Animal Drug Project of Gansu Province, Lanzhou 730050, China; (Y.Q.); (Y.B.); (W.W.); (Q.W.)
- Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture and Rural Affairs, Lanzhou 730050, China
- Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou 730050, China
- College of Veterinary Medicine, Northwest Agriculture & Forestry University, Yangling 712100, China
| | - Yubin Bai
- Key Laboratory of New Animal Drug Project of Gansu Province, Lanzhou 730050, China; (Y.Q.); (Y.B.); (W.W.); (Q.W.)
- Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture and Rural Affairs, Lanzhou 730050, China
- Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou 730050, China
| | - Weiwei Wang
- Key Laboratory of New Animal Drug Project of Gansu Province, Lanzhou 730050, China; (Y.Q.); (Y.B.); (W.W.); (Q.W.)
- Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture and Rural Affairs, Lanzhou 730050, China
- Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou 730050, China
| | - Qing Wang
- Key Laboratory of New Animal Drug Project of Gansu Province, Lanzhou 730050, China; (Y.Q.); (Y.B.); (W.W.); (Q.W.)
- Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture and Rural Affairs, Lanzhou 730050, China
- Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou 730050, China
| | - Shulin Chen
- College of Veterinary Medicine, Northwest Agriculture & Forestry University, Yangling 712100, China
| | - Jiyu Zhang
- Key Laboratory of New Animal Drug Project of Gansu Province, Lanzhou 730050, China; (Y.Q.); (Y.B.); (W.W.); (Q.W.)
- Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture and Rural Affairs, Lanzhou 730050, China
- Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou 730050, China
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Peng S, Ali Sabir I, Hu X, Chen J, Qin Y. Advancements in Reference Gene Selection for Fruit Trees: A Comprehensive Review. Int J Mol Sci 2024; 25:1142. [PMID: 38256212 PMCID: PMC10816256 DOI: 10.3390/ijms25021142] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2023] [Revised: 12/30/2023] [Accepted: 01/09/2024] [Indexed: 01/24/2024] Open
Abstract
Real-time quantitative polymerase chain reaction (qRT-PCR) has been widely used in gene expression analyses due to its advantages of sensitivity, accuracy and high throughput. The stability of internal reference genes has progressively emerged as a major factor affecting the precision of qRT-PCR results. However, the stability of the expression of the reference genes needs to be determined further in different cells or organs, physiological and experimental conditions. Methods for evaluating these candidate internal reference genes have also evolved from simple single software evaluation to more reliable and accurate internal reference gene evaluation by combining different software tools in a comprehensive analysis. This study intends to provide a definitive reference for upcoming research that will be conducted on fruit trees. The primary focus of this review is to summarize the research progress in recent years regarding the selection and stability analysis of candidate reference genes for different fruit trees.
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Affiliation(s)
- Shujun Peng
- Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables, College of Horticulture, South China Agricultural University, Guangzhou 510642, China; (S.P.); (X.H.); (J.C.)
- Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (South China), Ministry of Agriculture and Rural Affairs, College of Horticulture, South China Agricultural University, Guangzhou 510642, China;
| | - Irfan Ali Sabir
- Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (South China), Ministry of Agriculture and Rural Affairs, College of Horticulture, South China Agricultural University, Guangzhou 510642, China;
| | - Xinglong Hu
- Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables, College of Horticulture, South China Agricultural University, Guangzhou 510642, China; (S.P.); (X.H.); (J.C.)
- Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (South China), Ministry of Agriculture and Rural Affairs, College of Horticulture, South China Agricultural University, Guangzhou 510642, China;
| | - Jiayi Chen
- Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables, College of Horticulture, South China Agricultural University, Guangzhou 510642, China; (S.P.); (X.H.); (J.C.)
- Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (South China), Ministry of Agriculture and Rural Affairs, College of Horticulture, South China Agricultural University, Guangzhou 510642, China;
| | - Yonghua Qin
- Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables, College of Horticulture, South China Agricultural University, Guangzhou 510642, China; (S.P.); (X.H.); (J.C.)
- Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (South China), Ministry of Agriculture and Rural Affairs, College of Horticulture, South China Agricultural University, Guangzhou 510642, China;
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Mu D, Shao Y, He J, Zhu L, Qiu D, Wilson IW, Zhang Y, Pan L, Zhou Y, Lu Y, Tang Q. Evaluation of Reference Genes for Normalizing RT-qPCR and Analysis of the Expression Patterns of WRKY1 Transcription Factor and Rhynchophylline Biosynthesis-Related Genes in Uncaria rhynchophylla. Int J Mol Sci 2023; 24:16330. [PMID: 38003520 PMCID: PMC10671239 DOI: 10.3390/ijms242216330] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2023] [Revised: 11/10/2023] [Accepted: 11/13/2023] [Indexed: 11/26/2023] Open
Abstract
Uncaria rhynchophylla (Miq.) Miq. ex Havil, a traditional medicinal herb, is enriched with several pharmacologically active terpenoid indole alkaloids (TIAs). At present, no method has been reported that can comprehensively select and evaluate the appropriate reference genes for gene expression analysis, especially the transcription factors and key enzyme genes involved in the biosynthesis pathway of TIAs in U. rhynchophylla. Reverse transcription quantitative PCR (RT-qPCR) is currently the most common method for detecting gene expression levels due to its high sensitivity, specificity, reproducibility, and ease of use. However, this methodology is dependent on selecting an optimal reference gene to accurately normalize the RT-qPCR results. Ten candidate reference genes, which are homologues of genes used in other plant species and are common reference genes, were used to evaluate the expression stability under three stress-related experimental treatments (methyl jasmonate, ethylene, and low temperature) using multiple stability analysis methodologies. The results showed that, among the candidate reference genes, S-adenosylmethionine decarboxylase (SAM) exhibited a higher expression stability under the experimental conditions tested. Using SAM as a reference gene, the expression profiles of 14 genes for key TIA enzymes and a WRKY1 transcription factor were examined under three experimental stress treatments that affect the accumulation of TIAs in U. rhynchophylla. The expression pattern of WRKY1 was similar to that of tryptophan decarboxylase (TDC) under ETH treatment. This research is the first to report the stability of reference genes in U. rhynchophylla and provides an important foundation for future gene expression analyses in U. rhynchophylla. The RT-qPCR results indicate that the expression of WRKY1 is similar to that of TDC under ETH treatment. It may coordinate the expression of TDC, providing a possible method to enhance alkaloid production in the future through synthetic biology.
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Affiliation(s)
- Detian Mu
- College of Horticulture, Hunan Agricultural University, Changsha 410128, China
| | - Yingying Shao
- College of Horticulture, Hunan Agricultural University, Changsha 410128, China
| | - Jialong He
- College of Horticulture, Hunan Agricultural University, Changsha 410128, China
| | - Lina Zhu
- College of Horticulture, Hunan Agricultural University, Changsha 410128, China
| | - Deyou Qiu
- State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China
| | - Iain W Wilson
- Commonwealth Scientific and Industrial Research Organisation (CSIRO) Agriculture and Food, Canberra, ACT 2601, Australia
| | - Yao Zhang
- College of Horticulture, Hunan Agricultural University, Changsha 410128, China
| | - Limei Pan
- Key Laboratory of Guangxi for High-Quality Formation and Utilization of Dai-di Herbs, Guangxi Botanical Garden of Medicinal Plants, Nanning 530023, China
| | - Yu Zhou
- College of Horticulture, Hunan Agricultural University, Changsha 410128, China
| | - Ying Lu
- College of Horticulture, Hunan Agricultural University, Changsha 410128, China
| | - Qi Tang
- College of Horticulture, Hunan Agricultural University, Changsha 410128, China
- Hunan Provincial Key Laboratory for Synthetic Biology of Traditional Chinese Medicine, Hunan University of Medicine, Changsha 410208, China
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Jia X, Xiong Y, Xiong Y, Li D, Yu Q, Lei X, You M, Bai S, Zhang J, Ma X. Identification and Validation of Reference Genes for RT-qPCR Analysis in Reed Canary Grass during Abiotic Stress. Genes (Basel) 2023; 14:1790. [PMID: 37761930 PMCID: PMC10530813 DOI: 10.3390/genes14091790] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2023] [Revised: 09/07/2023] [Accepted: 09/08/2023] [Indexed: 09/29/2023] Open
Abstract
Reed canary grass (Phalaris arundinacea L.) is known for its tolerance to drought, heavy metals, and waterlogging, making it a popular choice for forage production and wetland restoration in the Qinghai-Tibet Plateau (QTP). To accurately assess gene expression in reed canary grass under different abiotic stresses, suitable reference genes need to be identified and validated. Thirteen candidate reference gene sequences were selected and screened using RT-qPCR to detect their expression levels in reed canary grass leaves under drought, salt, cadmium, and waterlogging stresses. Four algorithms were used to assess the stability of the expression levels of the candidate reference genes. The most stably expressed genes were UBC and H3 under drought Cd, ETF and CYT under salt stress, and ETF and TUB under waterlogging stress. GAPDH was found to be less stable under abiotic stresses. PIP-1, PAL, NAC 90, and WRKY 72A were selected as response genes for quantitative expression assessment under drought, salt, Cd, and waterlogging stresses to confirm the accuracy of the selected stable reference genes. These results provide a theoretical reference for assessing gene expression in reed canary grass under abiotic stresses.
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Affiliation(s)
- Xuejie Jia
- College of Grassland Science and Technology, Sichuan Agricultural University, Chengdu 611130, China; (X.J.)
- Sichuan Academy of Grassland Science, Chengdu 610097, China; (D.L.)
| | - Yi Xiong
- College of Grassland Science and Technology, Sichuan Agricultural University, Chengdu 611130, China; (X.J.)
| | - Yanli Xiong
- College of Grassland Science and Technology, Sichuan Agricultural University, Chengdu 611130, China; (X.J.)
| | - Daxu Li
- Sichuan Academy of Grassland Science, Chengdu 610097, China; (D.L.)
| | - Qinqin Yu
- Sichuan Academy of Grassland Science, Chengdu 610097, China; (D.L.)
| | - Xiong Lei
- Sichuan Academy of Grassland Science, Chengdu 610097, China; (D.L.)
| | - Minghong You
- Sichuan Academy of Grassland Science, Chengdu 610097, China; (D.L.)
| | - Shiqie Bai
- School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang 621002, China
| | - Jianbo Zhang
- Sichuan Academy of Grassland Science, Chengdu 610097, China; (D.L.)
| | - Xiao Ma
- College of Grassland Science and Technology, Sichuan Agricultural University, Chengdu 611130, China; (X.J.)
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Gomes TG, de Assis Fonseca FC, Alves GSC, de Siqueira FG, Miller RNG. Development of reference genes for RT-qPCR analysis of gene expression in Pleurotus pulmonarius for biotechnological applications. Sci Rep 2023; 13:12296. [PMID: 37516784 PMCID: PMC10387064 DOI: 10.1038/s41598-023-39115-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2023] [Accepted: 07/20/2023] [Indexed: 07/31/2023] Open
Abstract
Jatropha curcas is an oilseed crop with biorefinery applications. Whilst cake generated following oil extraction offers potential as a protein source for animal feed, inactivation of toxic phorbol esters present in the material is necessary. Pleurotus pulmonarius is a detoxifying agent for jatropha cake with additional potential as animal feed, edible mushroom and for enzyme production. For the characterization of fungal genes involved in phorbol ester degradation, together with other industrial applications, reverse transcription-quantitative PCR (RT-qPCR) is a tool that enables accurate quantification of gene expression. For this, reliable analysis requires reference genes for normalization of mRNA levels validated under conditions employed for target genes. The stability of potential reference genes β-TUB, ACTIN, GAPDH, PHOS, EF1α, TRPHO, LAC, MNP3, MYP and VP were evaluated following growth of P. pulmonarius on toxic, non-toxic jatropha cake and a combined treatment, respectively. NormFinder and geNorm algorithms for expression stability analysis identified PHOS, EF1α and MNP3 as appropriate for normalizing gene expression. Reference gene combinations contrasting in ranking were compared following normalization of relative expression of the CHU_2040 gene, encoding an esterase enzyme potentially involved in phorbol ester degradation. The reference genes for P. pulmonarius will facilitate the elucidation of mechanisms involved in detoxification of phorbol esters as well as analysis of target genes for application in biorefinery models.
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Affiliation(s)
- Taísa Godoy Gomes
- Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Campus Universitário Darcy Ribeiro, Brasília, DF, 70910-900, Brazil
| | - Fernando Campos de Assis Fonseca
- Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Campus Universitário Darcy Ribeiro, Brasília, DF, 70910-900, Brazil
- Instituto Federal de Goiás (IFG), Águas Lindas, GO, 72910-733, Brazil
| | - Gabriel Sergio Costa Alves
- Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Campus Universitário Darcy Ribeiro, Brasília, DF, 70910-900, Brazil
| | | | - Robert Neil Gerard Miller
- Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Campus Universitário Darcy Ribeiro, Brasília, DF, 70910-900, Brazil.
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Nevone A, Lattarulo F, Russo M, Panno G, Milani P, Basset M, Avanzini MA, Merlini G, Palladini G, Nuvolone M. A Strategy for the Selection of RT-qPCR Reference Genes Based on Publicly Available Transcriptomic Datasets. Biomedicines 2023; 11:1079. [PMID: 37189697 PMCID: PMC10135859 DOI: 10.3390/biomedicines11041079] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2023] [Revised: 03/23/2023] [Accepted: 03/27/2023] [Indexed: 04/05/2023] Open
Abstract
In the next-generation sequencing era, RT-qPCR is still widely employed to quantify levels of nucleic acids of interest due to its popularity, versatility, and limited costs. The measurement of transcriptional levels through RT-qPCR critically depends on reference genes used for normalization. Here, we devised a strategy to select appropriate reference genes for a specific clinical/experimental setting based on publicly available transcriptomic datasets and a pipeline for RT-qPCR assay design and validation. As a proof-of-principle, we applied this strategy to identify and validate reference genes for transcriptional studies of bone-marrow plasma cells from patients with AL amyloidosis. We performed a systematic review of published literature to compile a list of 163 candidate reference genes for RT-qPCR experiments employing human samples. Next, we interrogated the Gene Expression Omnibus to assess expression levels of these genes in published transcriptomic studies on bone-marrow plasma cells from patients with different plasma cell dyscrasias and identified the most stably expressed genes as candidate normalizing genes. Experimental validation on bone-marrow plasma cells showed the superiority of candidate reference genes identified through this strategy over commonly employed "housekeeping" genes. The strategy presented here may apply to other clinical and experimental settings for which publicly available transcriptomic datasets are available.
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Affiliation(s)
- Alice Nevone
- Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy
- Amyloidosis Research and Treatment Center, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy
| | - Francesca Lattarulo
- Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy
- Amyloidosis Research and Treatment Center, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy
| | - Monica Russo
- Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy
- Amyloidosis Research and Treatment Center, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy
| | - Giada Panno
- Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy
- Amyloidosis Research and Treatment Center, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy
| | - Paolo Milani
- Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy
- Amyloidosis Research and Treatment Center, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy
| | - Marco Basset
- Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy
- Amyloidosis Research and Treatment Center, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy
| | - Maria Antonietta Avanzini
- Pediatric Hematology Oncology, Cell Factory, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy
| | - Giampaolo Merlini
- Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy
- Amyloidosis Research and Treatment Center, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy
| | - Giovanni Palladini
- Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy
- Amyloidosis Research and Treatment Center, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy
| | - Mario Nuvolone
- Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy
- Amyloidosis Research and Treatment Center, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy
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10
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Mfsd2a attenuated hypoxic-ischemic brain damage via protection of the blood-brain barrier in mfat-1 transgenic mice. Cell Mol Life Sci 2023; 80:71. [PMID: 36820986 PMCID: PMC9950179 DOI: 10.1007/s00018-023-04716-9] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2022] [Revised: 01/10/2023] [Accepted: 02/01/2023] [Indexed: 02/24/2023]
Abstract
Previous studies have shown that mfat-1 transgenic mice have protective effects against some central nervous system (CNS) disorders, owing to the high docosahexaenoic acid (DHA) content enriched in their brains. However, whether this protective effect is connected to the blood-brain barrier (BBB) remains unclear. This study aims to investigate the mechanisms of the protective effect against hypoxic-ischemic brain damage (HIBD) of mfat-1 transgenic mice. mfat-1 mice not only demonstrated a significant amelioration of neurological dysfunction and neuronal damage but also partly maintained the physiological permeability of the BBB after HIBD. We initially showed this was associated with elevated major facilitator superfamily domain-containing 2a (Mfsd2a) expression on the BBB, resulting from more lysophosphatidylcholine (LPC)-DHA entering the brain. Wild-type (WT) mice showed a similar Mfsd2a expression trend after long-term feeding with an LPC-DHA-rich diet. Knockdown of Mfsd2a by siRNA intra-cerebroventricular (ICV) injection neutralized the protective effect against HIBD-induced BBB disruption in mfat-1 mice, further validating the protective function of Mfsd2a on BBB. HIBD-induced BBB high permeability was attenuated by Mfsd2a, primarily through a transcellular pathway to decrease caveolae-like vesicle-mediated transcytosis. Taken together, these findings not only reveal that mfat-1 transgenic mice have higher expression of Mfsd2a on the BBB, which partly sustains BBB permeability via vesicular transcytosis to alleviate the severity of HIBD, but also suggest that dietary intake of LPC-DHA may upregulate Mfsd2a expression as a novel therapeutic strategy for BBB dysfunction and survival in HIBD patients.
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11
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Wang Q, Guo C, Yang S, Zhong Q, Tian J. Screening and Verification of Reference Genes for Analysis of Gene Expression in Garlic ( Allium sativum L.) under Cold and Drought Stress. PLANTS (BASEL, SWITZERLAND) 2023; 12:763. [PMID: 36840111 PMCID: PMC9963267 DOI: 10.3390/plants12040763] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/22/2022] [Revised: 01/30/2023] [Accepted: 02/01/2023] [Indexed: 06/18/2023]
Abstract
The principal objective of this study was to screen and verify reference genes appropriate for gene expression evaluation during plant growth and development under distinct growth conditions. Nine candidate reference genes were screened based on garlic transcriptome sequence data. RT-qPCR was used to detect the expression levels of the aforementioned reference genes in specific tissues under drought and cold stress. Then, geNorm, NormFinder, BestKeeper, and ReFinder were used to consider the consistency of the expression levels of candidate reference genes. Finally, the stress-responsive gene expression of ascorbate peroxidase (APX) was quantitatively evaluated to confirm the chosen reference genes. Our results indicated that there were variations in the abundance and stability of nine reference gene transcripts underneath cold and drought stress, among which ACT and UBC-E2 had the highest transcript abundance, and 18S rRNA and HIS3 had the lowest transcript abundance. UBC and UBC-E2 were the most stably expressed genes throughout all samples; UBC and UBC-E2 were the most stably expressed genes during cold stress, and ACT and UBC were the most stably expressed genes under drought stress. The most stably expressed genes in roots, pseudostems, leaves, and cloves were EF1, ACT, HIS3, UBC, and UBC-E2, respectively, while GAPDH was the most unstable gene during drought and cold stress conditions and in exclusive tissues. Taking the steady reference genes UBC-E2, UBC, and ACT as references during drought and cold stress, the reliability of the expression levels was further demonstrated by detecting the expression of AsAPX. Our work thereby offers a theoretical reference for the evaluation of gene expression in garlic in various tissues and under stress conditions.
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Affiliation(s)
- Qizhang Wang
- Qinghai Key Laboratory of Vegetable Genetics and Physiology, Academy of Agriculture and Forestry Sciences of Qinghai University, Xining 810016, China
- School of Life Sciences, Lanzhou University, Lanzhou 730000, China
| | - Chunqian Guo
- Qinghai Key Laboratory of Vegetable Genetics and Physiology, Academy of Agriculture and Forestry Sciences of Qinghai University, Xining 810016, China
| | - Shipeng Yang
- Qinghai Key Laboratory of Vegetable Genetics and Physiology, Academy of Agriculture and Forestry Sciences of Qinghai University, Xining 810016, China
| | - Qiwen Zhong
- Qinghai Key Laboratory of Vegetable Genetics and Physiology, Academy of Agriculture and Forestry Sciences of Qinghai University, Xining 810016, China
| | - Jie Tian
- Qinghai Key Laboratory of Vegetable Genetics and Physiology, Academy of Agriculture and Forestry Sciences of Qinghai University, Xining 810016, China
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12
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Pant N, Rush C, Warner J, Eisen DP. Effect of Savirin or Ticagrelor Treatment on the Expression of Commonly Used Reference Genes in Staphylococcus aureus. Microorganisms 2023; 11:microorganisms11020336. [PMID: 36838300 PMCID: PMC9964243 DOI: 10.3390/microorganisms11020336] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2022] [Revised: 01/19/2023] [Accepted: 01/24/2023] [Indexed: 01/31/2023] Open
Abstract
Reference genes are frequently used for the normalization of quantitative reverse transcriptase PCR (qRTPCR) data in gene expression studies. Staphylococcus aureus is one of the most common causes of biofilm-related infections. Savirin and ticagrelor show in vitro as well as in vivo antibiofilm activity against S. aureus. The main aim of this study was to identify the most stably expressed reference genes to study the effect of these molecules on genes in a strong biofilm producing S. aureus isolate isolated from biofilm-related infection. Quantitative real-time PCR was performed by using relative quantification method. Four different algorithms, delta Ct, normfinder, bestkeeper, and genorm, followed by a comprehensive analysis was used to identify the most stable reference genes from a list of sixteen different candidate reference genes. All four algorithms reported different results, with some comparable findings among some methods. In the comprehensive analysis of the results of all the algorithms used, the most stable reference genes found were spa, rpoD, and pyk for savirin treatment experiment and gapdH, gyrA, and gmk for ticagrelor treatment experiment. The optimal number of reference genes required was two for both the experimental conditions. Despite having some drawbacks, each algorithm can reliably determine an appropriate reference gene independently. However, based on consensus ranking and the required optimal number of reference genes reported, spa and rpoD were the most appropriate reference genes for savirin treatment experiment, and gapdH and gyrA were most appropriate for ticagrelor treatment experiment. This study provides baseline data on reference genes to study the effect of savirin or ticagrelor treatment on the expression of potential reference genes in S. aureus. We recommend prior re-validation of reference genes on a case-by-case basis before they can be used.
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Affiliation(s)
- Narayan Pant
- College of Medicine and Dentistry, James Cook University, Townsville, QLD 4811, Australia
- Australian Institute of Tropical Health and Medicine, Townsville, QLD 4811, Australia
- Correspondence:
| | - Catherine Rush
- Australian Institute of Tropical Health and Medicine, Townsville, QLD 4811, Australia
| | - Jeffrey Warner
- Australian Institute of Tropical Health and Medicine, Townsville, QLD 4811, Australia
| | - Damon P. Eisen
- College of Medicine and Dentistry, James Cook University, Townsville, QLD 4811, Australia
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13
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Tani N, Ikeda T, Ishikawa T. Relationship between clock gene expression and CYP2C19 and CYP3A4 with benzodiazepines. Hum Exp Toxicol 2023; 42:9603271231171643. [PMID: 37072025 DOI: 10.1177/09603271231171643] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/20/2023]
Abstract
The present study aimed to clarify the expressions and roles of clock genes involved in drug metabolism in patients taking benzodiazepines (BZDs), as well as the drug metabolism regulators controlled by clock genes for each BZD type. The relationships between the expressions of the clock genes BMAL1, PER2, and DBP and the drug-metabolizing enzymes CYP3A4 and CYP2C19 were investigated using livers from BZD-detected autopsy cases. In addition, the effect of BZD exposure on various genes was examined in HepG2 human hepatocellular carcinoma cells. The expressions of DBP, CYP3A4, and CYP2C19 in the liver were lower in the diazepam-detected group than in the non-detected group. Furthermore, BMAL1 expression correlated with CYP2C19 expression. Cell culture experiments showed that the expressions of DBP and CYP3A4 decreased, whereas those of BMAL1 and CYP2C19 increased after diazepam and midazolam exposure. The results of the analyses of autopsy samples and cultured cells suggested that DBP regulates CYP3A4 when exposed to BZD. Understanding the relationship between these clock genes and CYPs may help achieve individualized drug therapy.
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Affiliation(s)
- Naoto Tani
- Department of Legal Medicine, Graduate School of Medicine, Osaka Metropolitan University, Abeno, Osaka, Japan
- Forensic Autopsy Section, Medico-legal Consultation and Postmortem Investigation Support Center, C/O Department of Legal Medicine, Graduate School of Medicine, Osaka Metropolitan University, Abeno, Osaka, Japan
| | - Tomoya Ikeda
- Department of Legal Medicine, Graduate School of Medicine, Osaka Metropolitan University, Abeno, Osaka, Japan
- Forensic Autopsy Section, Medico-legal Consultation and Postmortem Investigation Support Center, C/O Department of Legal Medicine, Graduate School of Medicine, Osaka Metropolitan University, Abeno, Osaka, Japan
| | - Takaki Ishikawa
- Department of Legal Medicine, Graduate School of Medicine, Osaka Metropolitan University, Abeno, Osaka, Japan
- Forensic Autopsy Section, Medico-legal Consultation and Postmortem Investigation Support Center, C/O Department of Legal Medicine, Graduate School of Medicine, Osaka Metropolitan University, Abeno, Osaka, Japan
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14
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Identification of RT-qPCR reference genes suitable for gene function studies in the pitaya canker disease pathogen Neoscytalidium dimidiatum. Sci Rep 2022; 12:22357. [PMID: 36572711 PMCID: PMC9792573 DOI: 10.1038/s41598-022-27041-w] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2022] [Accepted: 12/23/2022] [Indexed: 12/28/2022] Open
Abstract
Neoscytalidium dimidiatum is the main causal agent of pitaya canker. Most studies of virulence and pathogenicity genes have measured expression levels using real-time quantitative polymerase chain reaction (RT-qPCR). Suitable reference genes are essential for ensuring that estimates of gene expression levels by RT-qPCR are accurate. However, no reference genes can be robustly applied across all contexts and species. No studies to date have evaluated the most effective reference genes for normalizing gene expression levels estimated by RT-qPCR in N. dimidiatum. In this study, RT-qPCR data for individual candidate reference genes were analyzed using four different methods: the delta Ct method and the geNorm, NormFinder, and BestKeeper algorithms. We evaluated the utility of eight candidate reference genes (18S rRNA, Actin (1), Actin (2), Actin, GAPDH (1), GAPDH (2), UBQ, and Tubulin) for normalizing expression levels estimated by RT-qPCR in N. dimidiatum at different developmental stages, at different temperatures, and during interaction with pitaya. All candidate reference genes were suitable for gene expression analysis except for Actin (2). Tubulin and Actin (1) were the most stably expressed reference genes under different temperatures. Actin (1) and Actin were the most stably expressed reference genes in N. dimidiatum at different developmental stages. Tubulin and UBQ were the most stably expressed reference genes during interaction with pitaya. Actin and 18s rRNA were the most stably expressed across all experimental conditions. Subsequently, Tubulin and UBQ were further investigated in analyses of pectinase expression during the pitaya-N. dimidiatum interaction. Our results provide insights that will aid future RT-qPCR studies of gene expression in N. dimidiatum.
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15
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Evaluation of Reference Genes for Real-Time Quantitative PCR Analysis in Tissues from Bumble Bees ( Bombus Terrestris) of Different Lines. Int J Mol Sci 2022; 23:ijms232214371. [PMID: 36430847 PMCID: PMC9692494 DOI: 10.3390/ijms232214371] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2022] [Revised: 11/09/2022] [Accepted: 11/17/2022] [Indexed: 11/22/2022] Open
Abstract
Bumble bees are important alternative pollinators and model insects due to their highly developed sociality and colony management. In order to better understand their molecular mechanisms, studies focusing on the genetic and molecular aspects of their development and behavior are needed. Although quantitative real-time polymerase chain reaction (qRT-PCR) can be used to quantify the relative expression of target genes, internal reference genes (which are stably expressed across different lines and tissues) must first be identified to ensure the accurate normalization of target genes. In order to contribute to molecular studies on bumble bees, we used Bombus terrestris to determine the expression stability of eight reference genes (β-actin (ACT), Arginine Kinase (AK), Phospholipase A2 (PLA2), Elongation factor 1 alpha (EF-1), Ribosomal proteins (S5, S18, S28) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) in five different lines and several tissues (ovary, thorax, fat body, and head) using RT-qPCR procedures and four analysis programs (RefFinder, NormFinder, BestKeeper, and geNorm). In general, the S28, S5, and S18 ribosomal protein genes and the PLA2 and EF-1 genes showed the highest stability and were therefore identified as suitable reference genes for the bumble bee species and their defined lines and tissues. Our results also emphasized the need to evaluate the stability of candidate reference genes for any differently designed lines and tissue conditions in bumble bee species.
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16
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Selection of species specific panel of reference genes in peripheral blood mononuclear cells of native livestock species adapted to trans-Himalayan region of Leh-Ladakh. Sci Rep 2022; 12:18473. [PMID: 36323741 PMCID: PMC9630269 DOI: 10.1038/s41598-022-22588-0] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2021] [Accepted: 10/17/2022] [Indexed: 01/06/2023] Open
Abstract
The identification of appropriate references genes is an integral component of any gene expression-based study for getting accuracy and reliability in data interpretation. In this study, we evaluated the expression stability of 10 candidate reference genes (GAPDH, RPL4, EEF1A1, RPS9, HPRT1, UXT, RPS23, B2M, RPS15, ACTB) in peripheral blood mononuclear cells of livestock species that are adapted to high altitude hypoxia conditions of Leh-Ladakh. A total of 37 PBMCs samples from six native livestock species of Leh-Ladakh region such as Ladakhi cattle, Ladakhi yak, Ladakhi donkey, Chanthangi goat, Double hump cattle and Zanskar ponies were included in this study. The commonly used statistical algorithms such as geNorm, Normfinder, BestKeeper and RefFinder were employed to assess the stability of these RGs in all the livestock species. Our study has identified different panel of reference genes in each species; for example, EEF1A1, RPL4 in Ladakhi cattle; GAPDH, RPS9, ACTB in Ladakhi yak; HPRT1, B2M, ACTB in Ladakhi donkey; HPRT1, B2M, ACTB in Double hump camel, RPS9, HPRT1 in Changthangi goat, HPRT1 and ACTB in Zanskar ponies. To the best of our knowledge, this is the first systematic attempt to identify panel of RGs across different livestock species types adapted to high altitude hypoxia conditions. In future, the findings of the present study would be quite helpful in conducting any transcriptional studies to understand the molecular basis of high altitude adaptation of native livestock population of Leh-Ladakh.
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17
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Liu H, Lu Y, Wang X, Wang X, Li R, Lu C, Lan X, Chen Y. Selection and Validation of Reference Genes for RT-qPCR Analysis in Tibetan Medicinal Plant Saussurea Laniceps Callus under Abiotic Stresses and Hormone Treatments. Genes (Basel) 2022; 13:904. [PMID: 35627289 PMCID: PMC9140610 DOI: 10.3390/genes13050904] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2022] [Revised: 05/13/2022] [Accepted: 05/16/2022] [Indexed: 11/16/2022] Open
Abstract
Real-time quantitative PCR (RT-qPCR) is an important technique for studying gene expression analysis, but accurate and reliable results depend on the use of a stable reference gene. This study proposes to test the expression stability of candidate reference genes in the callus of Saussurea laniceps, a unique Tibetan medicinal plant. Based on the S. laniceps callus transcriptome, eleven candidate reference genes, including TUA2, TUB3, TUB8, TIF3B1, TIF3H1, ELF5A, PP2AA2, UEV1D, UBL5, UBC36, and SKIP1), were validated for RT-qPCR normalization in the callus under abiotic stress (salt, cold, and UV) and hormone treatments (abscisic acid, MeJA, and salicylic acid). The stability of the candidate genes was evaluated in all the samples of S. laniceps. Comprehensive analysis of all samples showed that the best reference genes were UBC36 and UBL5. ELF5A and TIF3B1 were ranked as the most stable genes in the sample sets under abiotic stress. For hormone stimulation, UBC36 and TIF3H1 genes had the best stability. This study provides useful guidelines and a starting point for reference gene selection for expression analysis using RT-qPCR techniques in S. laniceps.
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Affiliation(s)
- Huan Liu
- National Engineering Research Center of Tree Breeding and Ecological Restoration, Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education, The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of National Forestry and Grassland Administration, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China; (H.L.); (Y.L.); (X.W.); (R.L.); (C.L.)
| | - Yaning Lu
- National Engineering Research Center of Tree Breeding and Ecological Restoration, Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education, The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of National Forestry and Grassland Administration, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China; (H.L.); (Y.L.); (X.W.); (R.L.); (C.L.)
| | - Xiaojing Wang
- National Engineering Research Center of Tree Breeding and Ecological Restoration, Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education, The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of National Forestry and Grassland Administration, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China; (H.L.); (Y.L.); (X.W.); (R.L.); (C.L.)
| | - Xiaowei Wang
- Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China;
| | - Rongchen Li
- National Engineering Research Center of Tree Breeding and Ecological Restoration, Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education, The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of National Forestry and Grassland Administration, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China; (H.L.); (Y.L.); (X.W.); (R.L.); (C.L.)
| | - Cunfu Lu
- National Engineering Research Center of Tree Breeding and Ecological Restoration, Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education, The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of National Forestry and Grassland Administration, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China; (H.L.); (Y.L.); (X.W.); (R.L.); (C.L.)
| | - Xiaozhong Lan
- The Provincial and Ministerial Co-Founded Collaborative Innovation Center for R & D in Tibet Characteristic Agricultural and Animal Husbandry Resources, The Center for Xizang Chinese (Tibetan) Medicine Resource, Joint Laboratory for Tibetan Materia Medica Resources Scientific Protection and Utilization Research of Tibetan Medical Research Center of Tibet, Tibet Agriculture and Animal Husbandry University, Nyingchi 860000, China
| | - Yuzhen Chen
- National Engineering Research Center of Tree Breeding and Ecological Restoration, Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education, The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of National Forestry and Grassland Administration, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China; (H.L.); (Y.L.); (X.W.); (R.L.); (C.L.)
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18
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Tani N, Ikeda T, Ishikawa T. Effect of methamphetamine on clock genes and drug-metabolizing enzyme expression. Hum Exp Toxicol 2022; 41:9603271221124092. [PMID: 36036424 DOI: 10.1177/09603271221124092] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
This study examined the association between clock gene expression and the effect of methamphetamine (MA) on drug-metabolizing enzymes from the perspective of drug metabolism. The relationship between expression of the clock genes BMAL1 and PER2 and the drug-metabolizing enzymes CYP3A4 and CYP2D6 was investigated using livers from autopsy cases of MA-intoxication deaths. Additionally, the effect of MA exposure on various genes was examined in HepG2 human hepatocellular carcinoma cells. Comparisons of the expression of various genes in MA users according to blood MA concentration revealed that CYP3A4 expression was similar to that of PER2, and CYP2D6 expression was similar to that of BMAL1. In cultured cell experiments, BMAL1 and CYP2D6 expression decreased depending on the time elapsed after MA addition, and PER2 and CYP3A4 expression increased slightly in a concentration-dependent manner. These results were consistent with the findings of autopsy examinations. Expression of CYP3A4 and CYP2D6 under BMAL1 and PER2 suppression, but not CYP2D6 under PER2 suppression alone, was upregulated in response to MA. These results suggest that CYPs are regulated via the clock genes BMAL1 and PER2 during MA metabolism.
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Affiliation(s)
- Naoto Tani
- Department of Legal Medicine, Graduate School of Medicine, 12936Osaka Metropolitan University, Osaka, Japan.,Forensic Autopsy Section, Medico-legal Consultation and Postmortem Investigation Support Center, c/o Department of Legal Medicine, Graduate School of Medicine, 12936Osaka Metropolitan University, Osaka, Japan
| | - Tomoya Ikeda
- Department of Legal Medicine, Graduate School of Medicine, 12936Osaka Metropolitan University, Osaka, Japan.,Forensic Autopsy Section, Medico-legal Consultation and Postmortem Investigation Support Center, c/o Department of Legal Medicine, Graduate School of Medicine, 12936Osaka Metropolitan University, Osaka, Japan
| | - Takaki Ishikawa
- Department of Legal Medicine, Graduate School of Medicine, 12936Osaka Metropolitan University, Osaka, Japan.,Forensic Autopsy Section, Medico-legal Consultation and Postmortem Investigation Support Center, c/o Department of Legal Medicine, Graduate School of Medicine, 12936Osaka Metropolitan University, Osaka, Japan
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19
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Wu X, Yang W, Ren Z, Cheng J, Luo Y, Wang Y, Yang Z, Yao X, Zhao W, Li Y, Tang K. Reference gene screening for analyzing gene expression in the heart, liver, spleen, lung and kidney of forest musk deer. J Vet Med Sci 2021; 83:1750-1759. [PMID: 34615843 PMCID: PMC8636893 DOI: 10.1292/jvms.21-0281] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
The screening of reference genes for real-time quantitative PCR (qPCR) in forest musk deer (FMD) tissue is of great significance to the basic research on FMD. However, there are few reports
on the stability analysis of FMD reference genes so far. In this study, We used qPCR to detect the expression levels of 11 reference gene candidates (18S rRNA, beta-actin
[ACTB], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], TATA box-binding protein [TBP], hypoxanthine phosphoribosyltransferase 1
[HPRT1], tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide [YWHAZ], hydroxymethylbilane synthase
[HMBS], eukaryotic translation elongation factor 1 alpha 1 [EEF1A1], succinate dehydrogenase complex flavoprotein subunit A [SDHA],
peptidylprolyl isomerase B [PPIB], and ubiquitin C [UBC]) in heart, liver, spleen, lung and kidney of FMD. After removing 18S rRNA on
account of its high expression level, geNorm, NormFinder, BestKeeper and ΔCt algorithms were used to evaluate the expression stability of the remaining genes in the five organs, and further
comprehensive ranking was calculated by RefFinder. According to the results, the selected reference genes with the most stable expression in the heart of FMD are SDHA and
YWHAZ, while in the liver are ACTB and SDHA; in the spleen and lung are YWHAZ and HPRT1; in the kidney
are YWHAZ and PPIB. The use of common reference genes in all five organs is not recommended. The analyses showed that tissue is an important variability
factor in genes expression stability. Meanwhile, the result can be used as a reference for the selection of reference genes for qPCR in further study.
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Affiliation(s)
- Xi Wu
- College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, Sichuan Province, China
| | - Wei Yang
- College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, Sichuan Province, China
| | - Ziwei Ren
- College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, Sichuan Province, China
| | - Jianguo Cheng
- Sichuan Institute of Musk Deer Breeding, Dujiangyan 611830, Sichuan Province, China
| | - Yan Luo
- College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, Sichuan Province, China
| | - Yin Wang
- College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, Sichuan Province, China
| | - Zexiao Yang
- College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, Sichuan Province, China
| | - Xueping Yao
- College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, Sichuan Province, China
| | - Wei Zhao
- College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, Sichuan Province, China
| | - Yimeng Li
- College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, Sichuan Province, China
| | - Kexin Tang
- College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, Sichuan Province, China
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20
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Zhu Z, Gregg K, Zhou W. iRGvalid: A Robust in silico Method for Optimal Reference Gene Validation. Front Genet 2021; 12:716653. [PMID: 34422018 PMCID: PMC8372526 DOI: 10.3389/fgene.2021.716653] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2021] [Accepted: 07/19/2021] [Indexed: 11/24/2022] Open
Abstract
Background Appropriate reference genes are critical to accurately quantifying relative gene expression in research and clinical applications. Numerous efforts have been made to select the most stable reference gene(s), but a consensus has yet to be achieved. In this report, we propose an in silico reference gene validation method, iRGvalid, that can be used as a universal tool to validate the reference genes recommended from different resources so as to identify the best ones without a need for any wet lab validation tests. Methods iRGvalid takes advantage of high throughput gene expression data and is built on a double-normalization strategy. First, the expression level of each individual gene is normalized against the total gene expression level of each sample, followed by a target gene normalization to the candidate reference gene(s). Linear regression analysis is then performed between the pre- and post- normalized target gene across the whole sample set to evaluate the stability of the reference gene(s), which is positively associated with the Pearson correlation coefficient, Rt. The higher the Rt value, the more stable the reference gene. We applied iRGvalid to 14 candidate reference genes to validate and identify the most stable reference genes in four cancer types: lung adenocarcinoma, breast cancer, colon adenocarcinoma, and nasopharyngeal cancer. The stability of the reference gene is evaluated both individually and in groups of all possible combinations. Results Highly stable reference genes resulted in high Rt values regardless of the target gene used. The highest stability was achieved with a specific combination of 3 to 6 reference genes. A few genes were among the best reference genes across the cancer types studied here. Conclusion iRGvalid provides an easy and robust method to validate and identify the most stable reference gene or genes from a pool of candidate reference genes. The inclusivity of large expression data sets as well as the direct comparison of candidate reference genes makes it possible to identify reference genes with universal quality. This method can be used in any other gene expression studies when large cohorts of expression data are available.
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Affiliation(s)
| | | | - Wenli Zhou
- XYZ Laboratory, Austin, TX, United States
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21
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Lu X, Liu Y, Zhang D, Liu K, Wang Q, Wang H. Determination of the panel of reference genes for quantitative real-time PCR in fetal and adult rat intestines. Reprod Toxicol 2021; 104:68-75. [PMID: 34242779 DOI: 10.1016/j.reprotox.2021.07.001] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2021] [Revised: 06/30/2021] [Accepted: 07/04/2021] [Indexed: 02/07/2023]
Abstract
In quantitative real-time PCR (qRT-PCR) detection, the stability of reference genes varies with different organs, tissue locations, sex and developmental stages. This study aimed to screen out and determine the optimal panel of reference genes of the intestine in pre- and post-natal rats of different sex. We used qRT-PCR to detect the mRNA expression of six commonly used reference genes (ACTB, GAPDH, HPRT1, B2M, RPLPO and SDHA) in rat intestines at gestational day 21 (GD21) and postnatal week 12 (PW12). Using GeNorm, BestKeeper and NormFinder software comprehensively analyzed the stability of candidate reference genes and screened out stable reference genes. Further, we used the pathological model of prenatal dexamethasone exposure (PDE) to verify the stability of the selected panel of reference genes. Based on the results of the software analysis, the optimal panel of reference genes in the fetal rat intestine was SDHA + ACTB, and the adult rat small intestine and colon were ACTB + HPRT1 and RPLP0 + GAPDH, respectively. There was no significant sex difference in the above results. Besides, in the PDE model, the results were consistent with those under physiological conditions. Therefore, the stability of intestinal reference genes in fetal rats and adult rats was different, and the intestinal reference genes of adult rats were intestinal segments-specific. The selected panel of reference genes was still stable under pathological conditions. This study determined the optimal panel of reference genes of pre- and post-natal rat intestines and provided reliable reference genes for the qRT-PCR analysis of rat intestines.
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Affiliation(s)
- Xiaoqian Lu
- Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan, 430071, China
| | - Yi Liu
- Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan, 430071, China
| | - Dingmei Zhang
- Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan, 430071, China
| | - Kexin Liu
- Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan, 430071, China
| | - Qian Wang
- Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan, 430071, China
| | - Hui Wang
- Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan, 430071, China; Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan, 430071, China.
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22
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Reference gene selection for expression studies in the reproductive axis tissues of Magang geese at different reproductive stages under light treatment. Sci Rep 2021; 11:7573. [PMID: 33828187 PMCID: PMC8026621 DOI: 10.1038/s41598-021-87169-z] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2020] [Accepted: 03/19/2021] [Indexed: 11/09/2022] Open
Abstract
In quantitative PCR research, appropriate reference genes are key to determining accurate mRNA expression levels. In order to screen the reference genes suitable for detecting gene expression in tissues of the reproductive axis, a total of 420 (males and females = 1:5) 3-year-old Magang geese were selected and subjected to light treatment. The hypothalamus, pituitary and testicular tissues were subsequently collected at different stages. Ten genes including HPRT1, GAPDH, ACTB, LDHA, SDHA, B2M, TUBB4, TFRC, RPS2 and RPL4 were selected as candidate reference genes. The expression of these genes in goose reproductive axis tissues was detected by real-time fluorescent quantitative PCR. The ΔCT, geNorm, NormFinder and BestKeeper algorithms were applied to sort gene expression according to stability. The results showed that ACTB and TUBB4 were the most suitable reference genes for the hypothalamic tissue of Magang goose in the three breeding stages; HPRT1 and RPL4 for pituitary tissue; and HPRT1 and LDHA for testicular tissue. For all three reproductive axis tissues, ACTB was the most suitable reference gene, whereas the least stable reference gene was GAPDH. Altogether, these results can provide references for tissue expression studies in geese under light treatment.
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23
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Identification and validation of reference genes for reliable analysis of differential gene expression during antibiotic induced persister formation in Klebsiella pneumoniae using qPCR. J Microbiol Methods 2021; 182:106165. [PMID: 33581167 DOI: 10.1016/j.mimet.2021.106165] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2020] [Revised: 02/05/2021] [Accepted: 02/07/2021] [Indexed: 11/21/2022]
Abstract
The study of differential gene expression in persister cells is compounded by ceasure of conventional cellular metabolic pathways during persistence. There is, hence, a requirement to identify and validate suitable reference genes whose expression remains stable during persistence. We evaluated the suitability of five genes viz. dnaJ, groEL, rpoB, kp751, kp4432 as references to study gene expression using real-time polymerase chain reaction (qPCR) during persister cell formation in Klebsiella pneumoniae. Results obtained showed that while dnaJ and groEL suffered from unstable expression; rpoB, kp751 and kp4432 showed stable expression. Further, it was observed that data normalization using either of the stable genes viz. rpoB, kp751, kp4432 alone, resulted in either too low expression levels or too high variation among replicates. Our study indicates the concurrent use of kp4432 and rpoB as reference genes to be the most suitable for reliable analysis of differential gene expression during antibiotic induced persister formation in K. pneumoniae. kp4432 and rpoB encode NAD-dependant phenylacetaldehyde dehydrogenase and DNA-directed RNA polymerase beta subunit respectively. The outcome of this study will increase the utility of qPCR in studying the temporal changes in gene expression during persistence. The study will also aid in understanding mechanisms underlying persister cell formation particularly in K. pneumoniae.
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24
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Highet B, Vikas Anekal P, Ryan B, Murray H, Coppieters N, Victor Dieriks B, Singh-Bains MK, Mehrabi NF, Faull RLM, Dragunow M, Curtis MA. fISHing with immunohistochemistry for housekeeping gene changes in Alzheimer's disease using an automated quantitative analysis workflow. J Neurochem 2021; 157:1270-1283. [PMID: 33368239 DOI: 10.1111/jnc.15283] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2020] [Revised: 11/12/2020] [Accepted: 12/21/2020] [Indexed: 12/28/2022]
Abstract
In situ hybridization (ISH) is a powerful tool that can be used to localize mRNA expression in tissue samples. Combining ISH with immunohistochemistry (IHC) to determine cell type provides cellular context of mRNA expression, which cannot be achieved with gene microarray or polymerase chain reaction. To study mRNA and protein expression on the same section we investigated the use of RNAscope® ISH in combination with fluorescent IHC on paraffin-embedded human brain tissue. We first developed a high-throughput, automated image analysis workflow for quantifying RNA puncta across the total cell population and within neurons identified by NeuN+ immunoreactivity. We then applied this automated analysis to tissue microarray (TMA) sections of middle temporal gyrus tissue (MTG) from neurologically normal and Alzheimer's Disease (AD) cases to determine the suitability of three commonly used housekeeping genes: ubiquitin C (UBC), peptidyl-prolyl cis-trans isomerase B (PPIB) and DNA-directed RNA polymerase II subunit RPB1 (POLR2A). Overall, we saw a significant decrease in total and neuronal UBC expression in AD cases compared to normal cases. Total expression results were validated with RT-qPCR using fresh frozen tissue from 5 normal and 5 AD cases. We conclude that this technique combined with our novel automated analysis pipeline provides a suitable platform to study changes in gene expression in diseased human brain tissue with cellular and anatomical context. Furthermore, our results suggest that UBC is not a suitable housekeeping gene in the study of post-mortem AD brain tissue.
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Affiliation(s)
- Blake Highet
- Department of Anatomy and Medical Imaging, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand.,Centre for Brain Research, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
| | - Praju Vikas Anekal
- Biomedical Imaging Research Unit, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
| | - Brigid Ryan
- Department of Anatomy and Medical Imaging, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand.,Centre for Brain Research, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
| | - Helen Murray
- Department of Anatomy and Medical Imaging, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand.,Centre for Brain Research, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
| | - Natacha Coppieters
- Laboratory of Nervous System Disorders and Therapy, GIGA-Neuroscience, University of Liège, Liège, Belgium
| | - Birger Victor Dieriks
- Department of Anatomy and Medical Imaging, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand.,Centre for Brain Research, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
| | - Malvindar K Singh-Bains
- Department of Anatomy and Medical Imaging, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand.,Centre for Brain Research, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
| | - Nasim F Mehrabi
- Centre for Brain Research, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand.,Department of Pharmacology, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
| | - Richard L M Faull
- Department of Anatomy and Medical Imaging, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand.,Centre for Brain Research, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
| | - Michael Dragunow
- Centre for Brain Research, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand.,Department of Pharmacology, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
| | - Maurice A Curtis
- Department of Anatomy and Medical Imaging, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand.,Centre for Brain Research, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
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25
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Qin D, Zhao Y, Guo Q, Zhu S, Zhang S, Min L. Detection of Pancreatic Ductal Adenocarcinoma by A qPCR-based Normalizer-free Circulating Extracellular Vesicles RNA Signature. J Cancer 2021; 12:1445-1454. [PMID: 33531989 PMCID: PMC7847660 DOI: 10.7150/jca.50716] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2020] [Accepted: 10/25/2020] [Indexed: 12/17/2022] Open
Abstract
Background: Pancreatic ductal adenocarcinoma (PDAC) is difficult to diagnose and many efforts have been made to evaluate EVs-derived RNAs as biomarkers to predict PDAC. However, lack of robust internal references largely limited their clinical application. Here we proposed an RNA ratio-based, normalizer-free algorithm to quantitate EVs-derived RNAs in PDAC. Methods: Differentially expressed RNAs in the training group were identified using "limma" package. The ratio of any two candidate RNAs in the same sample was calculated and used as a new biomarker. LASSO regression was performed to build prediction models based on those RNA ratios. RNA-seq data of 116 plasma samples and RT-qPCR data of 111 plasma samples were used for internal and external validation, separately. Three algorithms (lasso regression, logistic regression, and SVM) were compared to improve the performance of this RNA signature. Results: We developed an RNA-ratio based prediction model which comprised eight EVs-derived RNAs, including FBXO7, MORF4L1, DDX17, TALDO1, AHNAK, TUBA1B, CD44, and SETD3. This model could well differentiate PDAC patients with a minimal AUC of 0.86 in internal verification using testing group. External validation using RT-qPCR data also exhibited a good classifier ability with an AUC of 0.89 when distinguishing PDAC from healthy controls. Conclusion: We've developed a qPCR-based, normalizer-free circulating EVs RNA classifier, which could well distinguish PDAC patients from noncancerous controls.
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Affiliation(s)
- Da Qin
- Department of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, National Clinical Research Center for Digestive Disease, Beijing Digestive Disease Center, Beijing Key Laboratory for Precancerous Lesion of Digestive Disease, Beijing, 100050, P. R. China
| | - Yu Zhao
- Department of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, National Clinical Research Center for Digestive Disease, Beijing Digestive Disease Center, Beijing Key Laboratory for Precancerous Lesion of Digestive Disease, Beijing, 100050, P. R. China
| | - Qingdong Guo
- Department of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, National Clinical Research Center for Digestive Disease, Beijing Digestive Disease Center, Beijing Key Laboratory for Precancerous Lesion of Digestive Disease, Beijing, 100050, P. R. China
| | - Shengtao Zhu
- Department of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, National Clinical Research Center for Digestive Disease, Beijing Digestive Disease Center, Beijing Key Laboratory for Precancerous Lesion of Digestive Disease, Beijing, 100050, P. R. China
| | - Shutian Zhang
- Department of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, National Clinical Research Center for Digestive Disease, Beijing Digestive Disease Center, Beijing Key Laboratory for Precancerous Lesion of Digestive Disease, Beijing, 100050, P. R. China
| | - Li Min
- Department of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, National Clinical Research Center for Digestive Disease, Beijing Digestive Disease Center, Beijing Key Laboratory for Precancerous Lesion of Digestive Disease, Beijing, 100050, P. R. China
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26
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Han H, Liu L, Chen M, Liu Y, Wang H, Chen L. The optimal compound reference genes for qRT-PCR analysis in the developing rat long bones under physiological conditions and prenatal dexamethasone exposure model. Reprod Toxicol 2020; 98:242-251. [DOI: 10.1016/j.reprotox.2020.10.008] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2020] [Revised: 10/10/2020] [Accepted: 10/14/2020] [Indexed: 10/23/2022]
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27
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Piccoli A, Cannata F, Strollo R, Pedone C, Leanza G, Russo F, Greto V, Isgrò C, Quattrocchi CC, Massaroni C, Silvestri S, Vadalà G, Bisogno T, Denaro V, Pozzilli P, Tang SY, Silva MJ, Conte C, Papalia R, Maccarrone M, Napoli N. Sclerostin Regulation, Microarchitecture, and Advanced Glycation End-Products in the Bone of Elderly Women With Type 2 Diabetes. J Bone Miner Res 2020; 35:2415-2422. [PMID: 32777114 PMCID: PMC8143610 DOI: 10.1002/jbmr.4153] [Citation(s) in RCA: 86] [Impact Index Per Article: 17.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/15/2020] [Revised: 07/21/2020] [Accepted: 08/02/2020] [Indexed: 12/21/2022]
Abstract
Increased circulating sclerostin and accumulation of advanced glycation end-products (AGEs) are two potential mechanisms underlying low bone turnover and increased fracture risk in type 2 diabetes (T2D). Whether the expression of the sclerostin-encoding SOST gene is altered in T2D, and whether it is associated with AGEs accumulation or regulation of other bone formation-related genes is unknown. We hypothesized that AGEs accumulate and SOST gene expression is upregulated in bones from subjects with T2D, leading to downregulation of bone forming genes (RUNX2 and osteocalcin) and impaired bone microarchitecture and strength. We obtained bone tissue from femoral heads of 19 T2D postmenopausal women (mean glycated hemoglobin [HbA1c] 6.5%) and 73 age- and BMI-comparable nondiabetic women undergoing hip replacement surgery. Despite similar bone mineral density (BMD) and biomechanical properties, we found a significantly higher SOST (p = .006) and a parallel lower RUNX2 (p = .025) expression in T2D compared with non-diabetic subjects. Osteocalcin gene expression did not differ between T2D and non-diabetic subjects, as well as circulating osteocalcin and sclerostin levels. We found a 1.5-fold increase in total bone AGEs content in T2D compared with non-diabetic women (364.8 ± 78.2 versus 209.9 ± 34.4 μg quinine/g collagen, respectively; p < .001). AGEs bone content correlated with worse bone microarchitecture, including lower volumetric BMD (r = -0.633; p = .02), BV/TV (r = -0.59; p = .033) and increased trabecular separation/spacing (r = 0.624; p = .023). In conclusion, our data show that even in patients with good glycemic control, T2D affects the expression of genes controlling bone formation (SOST and RUNX2). We also found that accumulation of AGEs is associated with impaired bone microarchitecture. We provide novel insights that may help understand the mechanisms underlying bone fragility in T2D. © 2020 American Society for Bone and Mineral Research (ASBMR).
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Affiliation(s)
- Alessandra Piccoli
- Unit of Endocrinology and Diabetes, Departmental Faculty of Medicine and Surgery, Campus Bio-Medico University of Rome, Rome, Italy.,Unit of Biochemistry and Molecular Biology, Departmental Faculty of Medicine and Surgery, Campus Bio-Medico University of Rome, Rome, Italy
| | - Francesca Cannata
- Unit of Endocrinology and Diabetes, Departmental Faculty of Medicine and Surgery, Campus Bio-Medico University of Rome, Rome, Italy
| | - Rocky Strollo
- Unit of Endocrinology and Diabetes, Departmental Faculty of Medicine and Surgery, Campus Bio-Medico University of Rome, Rome, Italy
| | - Claudio Pedone
- Unit of Geriatrics, Departmental Faculty of Medicine and Surgery, Campus Bio-Medico University of Rome, Rome, Italy
| | - Giulia Leanza
- Unit of Endocrinology and Diabetes, Departmental Faculty of Medicine and Surgery, Campus Bio-Medico University of Rome, Rome, Italy
| | - Fabrizio Russo
- Unit of Orthopedic and Trauma Surgery, Departmental Faculty of Medicine and Surgery, Campus Bio-Medico University of Rome, Rome, Italy
| | - Valentina Greto
- Unit of Endocrinology and Diabetes, Departmental Faculty of Medicine and Surgery, Campus Bio-Medico University of Rome, Rome, Italy
| | - Camilla Isgrò
- Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari "Aldo Moro", Bari, Italy
| | | | - Carlo Massaroni
- Research Unit of Measurements and Biomedical Instrumentation, Departmental Faculty of Bioengineering, Campus Bio-Medico di Roma University, Rome, Italy
| | - Sergio Silvestri
- Research Unit of Measurements and Biomedical Instrumentation, Departmental Faculty of Bioengineering, Campus Bio-Medico di Roma University, Rome, Italy
| | - Gianluca Vadalà
- Unit of Orthopedic and Trauma Surgery, Departmental Faculty of Medicine and Surgery, Campus Bio-Medico University of Rome, Rome, Italy
| | - Tiziana Bisogno
- Endocannabinoid Research Group, Institute of Translational Pharmacology, National Research Council, (CNR), Rome, Italy
| | - Vincenzo Denaro
- Unit of Orthopedic and Trauma Surgery, Departmental Faculty of Medicine and Surgery, Campus Bio-Medico University of Rome, Rome, Italy
| | - Paolo Pozzilli
- Unit of Endocrinology and Diabetes, Departmental Faculty of Medicine and Surgery, Campus Bio-Medico University of Rome, Rome, Italy
| | - Simon Y Tang
- Unit of Orthopedics, Washington University in St. Louis, St. Louis, MO, USA
| | - Matt J Silva
- Unit of Orthopedics, Washington University in St. Louis, St. Louis, MO, USA
| | - Caterina Conte
- Department of Human Sciences and Promotion of the Quality of Life, San Raffaele Roma Open University, Rome, Italy
| | - Rocco Papalia
- Unit of Orthopedic and Trauma Surgery, Departmental Faculty of Medicine and Surgery, Campus Bio-Medico University of Rome, Rome, Italy
| | - Mauro Maccarrone
- Unit of Biochemistry and Molecular Biology, Departmental Faculty of Medicine and Surgery, Campus Bio-Medico University of Rome, Rome, Italy.,European Center for Brain Research (CERC)/Santa Lucia Foundation, Rome, Italy
| | - Nicola Napoli
- Unit of Endocrinology and Diabetes, Departmental Faculty of Medicine and Surgery, Campus Bio-Medico University of Rome, Rome, Italy.,Division of Bone and Mineral Diseases, Washington University in St. Louis, St. Louis, MO, USA
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Differences in sphere-forming cells from keratoconic and normal corneal tissue: Implications for keratoconus pathogenesis. Exp Eye Res 2020; 202:108301. [PMID: 33086037 DOI: 10.1016/j.exer.2020.108301] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2020] [Revised: 09/15/2020] [Accepted: 10/12/2020] [Indexed: 11/23/2022]
Abstract
Keratoconus is primarily an anterior corneal disorder of unclear aetiology. Stem cells may play a role in the perpetuation of keratoconus, although this has yet to be definitively established. Sphere-forming cells from normal human donor corneas have previously been shown to be a heterogenous mix of epithelial, stromal, stem and progenitor cell components which have potential for treatment of corneal dystrophies. Our work set out to isolate and characterise sphere-forming cells from human keratoconic tissue. Keratoconic donor corneas were successfully used to culture sphere-forming cells in vitro. Time lapse imaging of these spheres on a collagen surface over 8 days revealed keratoconic spheres lack the ability to maintain a central core and have diminished ability to repopulate the surface. Immunocytochemistry showed positive labelling for the stem cell marker 'Adenosine triphosphate-binding cassette sub-family B member 5 (ABCB5)' indicating stem cell retention and the myofibroblast marker alpha smooth muscle actin indicating wound repair while droplet digital Polymerase Chain Reaction confirmed an increase in expression of stem and stromal cell markers in keratoconic spheres compared to spheres cultured from normal donors at day 7 post-placement. Keratoconic sphere-forming cells showed a diminished repopulation ability, a faster wound healing response and lack of central core retention. These results suggest stem cells in keratoconus may be in an elevated state of wound repair and unable to respond appropriately to further injury in corneal maintenance. Sphere forming cell populations in keratoconus appear to be different to those isolated from normal corneas and this may be an important consideration in unearthing keratoconus aetiology.
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Maia M, Ferreira AEN, Nascimento R, Monteiro F, Traquete F, Marques AP, Cunha J, Eiras-Dias JE, Cordeiro C, Figueiredo A, Sousa Silva M. Integrating metabolomics and targeted gene expression to uncover potential biomarkers of fungal/oomycetes-associated disease susceptibility in grapevine. Sci Rep 2020; 10:15688. [PMID: 32973337 PMCID: PMC7515887 DOI: 10.1038/s41598-020-72781-2] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2020] [Accepted: 09/02/2020] [Indexed: 12/20/2022] Open
Abstract
Vitis vinifera, one of the most cultivated fruit crops, is susceptible to several diseases particularly caused by fungus and oomycete pathogens. In contrast, other Vitis species (American, Asian) display different degrees of tolerance/resistance to these pathogens, being widely used in breeding programs to introgress resistance traits in elite V. vinifera cultivars. Secondary metabolites are important players in plant defence responses. Therefore, the characterization of the metabolic profiles associated with disease resistance and susceptibility traits in grapevine is a promising approach to identify trait-related biomarkers. In this work, the leaf metabolic composition of eleven Vitis genotypes was analysed using an untargeted metabolomics approach. A total of 190 putative metabolites were found to discriminate resistant/partial resistant from susceptible genotypes. The biological relevance of discriminative compounds was assessed by pathway analysis. Several compounds were selected as promising biomarkers and the expression of genes coding for enzymes associated with their metabolic pathways was analysed. Reference genes for these grapevine genotypes were established for normalisation of candidate gene expression. The leucoanthocyanidin reductase 2 gene (LAR2) presented a significant increase of expression in susceptible genotypes, in accordance with catechin accumulation in this analysis group. Up to our knowledge this is the first time that metabolic constitutive biomarkers are proposed, opening new insights into plant selection on breeding programs.
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Affiliation(s)
- Marisa Maia
- Laboratório de FTICR e Espectrometria de Massa Estrutural, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016, Lisbon, Portugal
- Biosystems and Integrative Sciences Institute (BioISI), Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016, Lisbon, Portugal
| | - António E N Ferreira
- Laboratório de FTICR e Espectrometria de Massa Estrutural, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016, Lisbon, Portugal
| | - Rui Nascimento
- Biosystems and Integrative Sciences Institute (BioISI), Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016, Lisbon, Portugal
| | - Filipa Monteiro
- Centre for Ecology, Evolution and Environmental Changes (cE3c), Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016, Lisbon, Portugal
- Linking Landscape, Environment, Agriculture and Food (LEAF), Instituto Superior de Agronomia (ISA), Universidade de Lisboa, 1349-017, Lisbon, Portugal
| | - Francisco Traquete
- Laboratório de FTICR e Espectrometria de Massa Estrutural, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016, Lisbon, Portugal
| | - Ana P Marques
- Laboratório de FTICR e Espectrometria de Massa Estrutural, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016, Lisbon, Portugal
| | - Jorge Cunha
- Instituto Nacional de Investigação Agrária e Veterinária (INIAV), Quinta da Almoinha, 2565-191, Dois Portos, Portugal
| | - José E Eiras-Dias
- Instituto Nacional de Investigação Agrária e Veterinária (INIAV), Quinta da Almoinha, 2565-191, Dois Portos, Portugal
| | - Carlos Cordeiro
- Laboratório de FTICR e Espectrometria de Massa Estrutural, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016, Lisbon, Portugal
| | - Andreia Figueiredo
- Biosystems and Integrative Sciences Institute (BioISI), Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016, Lisbon, Portugal.
| | - Marta Sousa Silva
- Laboratório de FTICR e Espectrometria de Massa Estrutural, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016, Lisbon, Portugal.
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Liu Y, Liu M, Zhang Q, Chen G, Wang H. Selection and identification of the panel of reference genes for quantitative real-time PCR normalization in rat testis at different development periods. Toxicol Appl Pharmacol 2020; 406:115243. [PMID: 32949581 DOI: 10.1016/j.taap.2020.115243] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2020] [Revised: 08/30/2020] [Accepted: 09/13/2020] [Indexed: 11/28/2022]
Abstract
BACKGROUND In quantitative real-time PCR (qRT-PCR), the expression levels of various adult reference genes may be unstable at different developmental periods and tissues, and will lead to inaccurate detected results. This study aimed to select and identify the optimal panel of reference genes in rat testis at different development periods. METHODS We detected mRNA expression levels of five common rat testicular reference genes (GAPDH, β-actin, 18s, RPS16 and RPL19) by qRT-PCR at different developmental periods (fetus, infancy, and adolescence), selected optimal panel of reference genes by combining with stability algorithms, and verified their tissue specificity. Lastly, we observed their mRNA expression alterations under pathological conditions to evaluate the stability and accuracy, and verify testicular dysplasia model. RESULTS Based on comprehensive analysis, the best panel of reference genes of testis were GAPDH+β-actin (at fetus) and GAPDH+RPL19 (at infancy and adolescent). Meanwhile, the best panel of reference genes of fetal testis was consistent with placenta and fetal hippocampus but different from fetal liver and kidney. Furthermore, in prenatal dexamethasone exposure (PDE) model, the results were consistent with those under physiological conditions, and the testicular steroidogenesis acute regulatory protein (StAR) was most obviously decreased when using the best panel of reference genes. CONCLUSION In this study, rat testicular GAPDH+β-actin for fetuses and GAPDH+RPL19 for infants and adolescents are recommended to be the optimal panel of reference genes. Respectively. The selected panel of reference genes was still stable under PDE condition. This study provided technical and theoretical supports for researches on testicular development toxicology.
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Affiliation(s)
- Yi Liu
- Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan 430071, China
| | - Min Liu
- Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan 430071, China
| | - Qi Zhang
- Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan 430071, China
| | - Guanghui Chen
- Department of Pharmacology, Wuhan University School of Basic Medical Sciences, Wuhan 430071, China
| | - Hui Wang
- Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan 430071, China; Department of Pharmacology, Wuhan University School of Basic Medical Sciences, Wuhan 430071, China.
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Hul LM, Ibelli AMG, Peixoto JDO, Souza MR, Savoldi IR, Marcelino DEP, Tremea M, Ledur MC. Reference genes for proximal femoral epiphysiolysis expression studies in broilers cartilage. PLoS One 2020; 15:e0238189. [PMID: 32841273 PMCID: PMC7447007 DOI: 10.1371/journal.pone.0238189] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2020] [Accepted: 08/11/2020] [Indexed: 12/18/2022] Open
Abstract
The use of reference genes is required for relative quantification in gene expression analysis and the stability of these genes can be variable depending on the experimental design. Therefore, it is indispensable to test the reliability of endogenous genes previously to their use. This study evaluated nine candidate reference genes to select the most stable genes to be used as reference in gene expression studies with the femoral cartilage of normal and epiphysiolysis-affected broilers. The femur articular cartilage of 29 male broilers with 35 days of age was collected, frozen and further submitted to RNA extraction and quantitative PCR (qPCR) analysis. The candidate reference genes evaluated were GAPDH, HMBS, HPRT1, MRPS27, MRPS30, RPL30, RPL4, RPL5, and RPLP1. For the gene stability evaluation, three software were used: GeNorm, BestKeeper and NormFinder, and a global ranking was generated using the function RankAggreg. In this study, the RPLP1 and RPL5 were the most reliable endogenous genes being recommended for expression studies with femur cartilage in broilers with epiphysiolysis and possible other femur anomalies.
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Affiliation(s)
- Ludmila Mudri Hul
- Programa de Pós-Graduação em Ciências Veterinárias, Universidade Estadual do Centro-Oeste, Guarapuava, Paraná, Brazil
| | - Adriana Mércia Guaratini Ibelli
- Programa de Pós-Graduação em Ciências Veterinárias, Universidade Estadual do Centro-Oeste, Guarapuava, Paraná, Brazil
- Embrapa Suínos e Aves, Concórdia, Santa Catarina, Brazil
| | - Jane de Oliveira Peixoto
- Programa de Pós-Graduação em Ciências Veterinárias, Universidade Estadual do Centro-Oeste, Guarapuava, Paraná, Brazil
- Embrapa Suínos e Aves, Concórdia, Santa Catarina, Brazil
| | - Mayla Regina Souza
- Programa de Pós-Graduação em Zootecnia, UDESC-Oeste, Chapecó, Santa Catarina, Brazil
| | - Igor Ricardo Savoldi
- Programa de Pós-Graduação em Zootecnia, UDESC-Oeste, Chapecó, Santa Catarina, Brazil
| | | | - Mateus Tremea
- Universidade Federal de Santa Maria, campus Palmeira das Missões, Rio Grande do Sul, Brazil
| | - Mônica Corrêa Ledur
- Embrapa Suínos e Aves, Concórdia, Santa Catarina, Brazil
- Programa de Pós-Graduação em Zootecnia, UDESC-Oeste, Chapecó, Santa Catarina, Brazil
- * E-mail:
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Selection and Validation of Reference Genes for Quantitative Real-Time PCR in White Clover ( Trifolium repens L.) Involved in Five Abiotic Stresses. PLANTS 2020; 9:plants9080996. [PMID: 32764378 PMCID: PMC7463471 DOI: 10.3390/plants9080996] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/08/2020] [Revised: 08/01/2020] [Accepted: 08/02/2020] [Indexed: 01/28/2023]
Abstract
White clover (Trifolium repens L.) is a widely cultivated cool-season perennial forage legume in temperate grassland systems. Many studies have analyzed the gene expression in this grass species using quantitative real-time reverse transcription PCR (qRT-PCR). The selection of stable reference genes for qRT-PCR is crucial. However, there was no detailed study on reference genes in different tissues of white clover under various abiotic stress conditions. Herein, 14 candidate reference genes (ACT7, ACT101, TUA1109, TUB, CYP, 60SrRNA, UBQ, E3, GAPDH1, GAPDH2, PP2A, BAM3, SAMDC, and ABC) were selected and analyzed by four programs (GeNorm, NormFinder, BestKeeper, and RefFinder). Samples were taken from two tissues (leaves and roots) under five different abiotic stresses (drought, salt, heat, cold, and heavy metal stress). Our results showed that 60SrRNA and ACT101 were the two top-ranked genes for all samples. Under various experimental conditions, the most stable gene was different; however, SAMDC, UBQ, 60SrRNA, and ACT101 were always top ranked. The most suitable reference genes should be selected according to different plant tissues and growth conditions. Validation of these reference genes by expression analysis of Cyt-Cu/Zn SOD and CAT confirmed their reliability. Our study will benefit the subsequent research of gene function in this species.
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Hromádková J, Suzuki Y, Pletts S, Pyo J, Ma T, Chen Y, Steele MA, Guan LL. Effect of colostrum feeding strategies on the expression of neuroendocrine genes and active gut mucosa-attached bacterial populations in neonatal calves. J Dairy Sci 2020; 103:8629-8642. [PMID: 32622610 DOI: 10.3168/jds.2019-17710] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2019] [Accepted: 04/21/2020] [Indexed: 01/10/2023]
Abstract
Colostrum feeding is vital for the development of the immune system and gastrointestinal tract in neonatal calves; however, it is currently unknown whether different colostrum feeding strategies affect their neuroendocrine system and potentially the gut-brain axis. The present study investigated the effect of 3 different colostrum feeding regimens on the expression of neuroendocrine genes in adrenal glands and gastrointestinal tissues and on the abundance of intestinal commensal bacteria. Holstein bull calves were fed colostrum immediately after birth and randomly assigned to 3 groups: whole milk (n = 8), mixture of 50% colostrum and 50% whole milk (n = 8), and colostrum (CF; n = 8) for 72 h with 12-h intervals. Adrenal glands, ileum, and colon tissues were collected at 75 h and were subjected to the expression of 11 targeted neuroendocrine genes and the abundance of tissue mucosa-associated bacteria measurement using quantitative real-time PCR and quantitative PCR, respectively. The expressions of all targeted genes were detected, and the expression of α-adrenergic receptor (ADRA1A) gene was affected by CF in adrenal glands and gut tissues. In addition, CF upregulated the expression of HTR4 (serotonin receptor) and SLC4A4 (serotonin transporter) genes in the ileum and increased the abundance of active Lactobacillus spp. and Escherichia coli (as detected at RNA level) associated with ileum and colon tissue. Furthermore, there were positive correlations between the abundance of active Lactobacillus spp. and E. coli with expression of HTR2B and HTR4 genes in the colon, suggesting that extended colostrum feeding strategies may affect the interaction between gut microbiota and host endocrine functions in neonatal calves.
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Affiliation(s)
- Jitka Hromádková
- Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5
| | - Yutaka Suzuki
- Laboratory of Animal Function and Nutrition, Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan 060-8589
| | - Sarah Pletts
- Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5
| | - Jade Pyo
- Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5
| | - Tao Ma
- Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5; Key Laboratory of Feed Biotechnology of the Ministry of Agriculture and Rural Affairs, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China 100081
| | - Yanhong Chen
- Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5
| | - Michael A Steele
- Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5; Department of Animal Biosciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1
| | - Le Luo Guan
- Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5.
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Zottel A, Jovčevska I, Šamec N, Mlakar J, Šribar J, Križaj I, Skoblar Vidmar M, Komel R. Anti-vimentin, anti-TUFM, anti-NAP1L1 and anti-DPYSL2 nanobodies display cytotoxic effect and reduce glioblastoma cell migration. Ther Adv Med Oncol 2020; 12:1758835920915302. [PMID: 32426045 PMCID: PMC7222267 DOI: 10.1177/1758835920915302] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2019] [Accepted: 03/04/2020] [Indexed: 11/17/2022] Open
Abstract
Background: Glioblastoma is a particularly common and very aggressive primary brain tumour. One of the main causes of therapy failure is the presence of glioblastoma stem cells that are resistant to chemotherapy and radiotherapy, and that have the potential to form new tumours. This study focuses on validation of eight novel antigens, TRIM28, nucleolin, vimentin, nucleosome assembly protein 1-like 1 (NAP1L1), mitochondrial translation elongation factor (EF-TU) (TUFM), dihydropyrimidinase-related protein 2 (DPYSL2), collapsin response mediator protein 1 (CRMP1) and Aly/REF export factor (ALYREF), as putative glioblastoma targets, using nanobodies. Methods: Expression of these eight antigens was analysed at the cellular level by qPCR, ELISA and immunocytochemistry, and in tissues by immunohistochemistry. The cytotoxic effects of the nanobodies were determined using AlamarBlue and water-soluble tetrazolium tests. Annexin V/propidium iodide tests were used to determine apoptotsis/necrosis of the cells in the presence of the nanobodies. Cell migration assays were performed to determine the effects of the nanobodies on cell migration. Results: NAP1L1 and CRMP1 were significantly overexpressed in glioblastoma stem cells in comparison with astrocytes and glioblastoma cell lines at the mRNA and protein levels. Vimentin, DPYSL2 and ALYREF were overexpressed in glioblastoma cell lines only at the protein level. The functional part of the study examined the cytotoxic effects of the nanobodies on glioblastoma cell lines. Four of the nanobodies were selected in terms of their specificity towards glioblastoma cells and protein overexpression: anti-vimentin (Nb79), anti-NAP1L1 (Nb179), anti-TUFM (Nb225) and anti-DPYSL2 (Nb314). In further experiments to optimise the nanobody treatment schemes, to increase their effects, and to determine their impact on migration of glioblastoma cells, the anti-TUFM nanobody showed large cytotoxic effects on glioblastoma stem cells, while the anti-vimentin, anti-NAP1L1 and anti-DPYSL2 nanobodies were indicated as agents to target mature glioblastoma cells. The anti-vimentin nanobody also had significant effects on migration of mature glioblastoma cells. Conclusion: Nb79 (anti-vimentin), Nb179 (anti-NAP1L1), Nb225 (anti-TUFM) and Nb314 (anti-DPYSL2) nanobodies are indicated for further examination for cell targeting. The anti-TUFM nanobody, Nb225, is particularly potent for inhibition of cell growth after long-term exposure of glioblastoma stem cells, with minor effects seen for astrocytes. The anti-vimentin nanobody represents an agent for inhibition of cell migration.
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Affiliation(s)
- Alja Zottel
- Medical Centre for Molecular Biology, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
| | - Ivana Jovčevska
- Medical Centre for Molecular Biology, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
| | - Neja Šamec
- Medical Centre for Molecular Biology, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
| | - Jernej Mlakar
- Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
| | - Jernej Šribar
- Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Ljubljana, Slovenia
| | - Igor Križaj
- Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Ljubljana, Slovenia
| | | | - Radovan Komel
- Medical Centre for Molecular Biology, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000, Ljubljana, Slovenia
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Zhang K, Cheng M, Xu J, Chen L, Li J, Li Q, Xie X, Wang Q. MiR-711 and miR-183-3p as potential markers for vital reaction of burned skin. Forensic Sci Res 2020; 7:503-509. [DOI: 10.1080/20961790.2020.1719454] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022] Open
Affiliation(s)
- Kaikai Zhang
- Department of Forensic Pathology, School of Forensic Medicine, Southern Medical University, Guangzhou, China
| | - Ming Cheng
- Forensic Science Centre of Guangdong Provincial Public Security Department, Guangzhou, China
| | - Jingtao Xu
- Department of Forensic Pathology, School of Forensic Medicine, Southern Medical University, Guangzhou, China
| | - Lijian Chen
- Department of Forensic Pathology, School of Forensic Medicine, Southern Medical University, Guangzhou, China
| | - Jiahao Li
- Department of Forensic Pathology, School of Forensic Medicine, Southern Medical University, Guangzhou, China
| | - Qiangguo Li
- Department of Critical Medicine, Mudan District People’s Hospital, Heze, China
| | - Xiaoli Xie
- Department of Toxicology, School of Public Health, Southern Medical University, Guangzhou, China
| | - Qi Wang
- Department of Forensic Pathology, School of Forensic Medicine, Southern Medical University, Guangzhou, China
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Qu D, Tan XH, Zhang KK, Wang Q, Wang HJ. ATF3 mRNA, but not BTG2, as a possible marker for vital reaction of skin contusion. Forensic Sci Int 2019; 303:109937. [PMID: 31546162 DOI: 10.1016/j.forsciint.2019.109937] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2018] [Revised: 08/08/2019] [Accepted: 08/22/2019] [Indexed: 11/18/2022]
Abstract
The detection of vitality of wounds, especially when the wounds are inflicted very close to the time of death, is one of the most challenging issues in forensic pathology. This study investigated expression levels of ATF3 and BTG2 in mouse and human skin wounds. Protein levels examined by western blot showed that there was no significant change in ATF3 and BTG2 between wounded and intact skins. However, mRNA levels demonstrated higher expression of ATF3 and BTG2 in ante-mortem contused mouse skins, compared with the intact and postmortem contused skins. Increased ATF3 and BTG2 in the level of mRNA could also be detected until 96h and 48h after death, respectively. Human wounded skin samples from forensic autopsy cases were also examined. Increased ATF3 mRNA levels were detected until 48h after autopsy in 5 of 6 cases. However, no differences were observed between wounded and intact skins for BTG2. These findings suggest that the detection of mRNA levels of ATF3, but not BTG2, can be considered as a potential marker for vital reaction of skin contusion. Postmortem human samples should be used in order to validate the availability of markers screened by animal experiment.
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Affiliation(s)
- Dong Qu
- Department of Forensic Pathology, School of Forensic Medicine, Southern Medical University, Guangzhou, China
| | - Xiao-Hui Tan
- Department of Forensic Pathology, School of Forensic Medicine, Southern Medical University, Guangzhou, China
| | - Kai-Kai Zhang
- Department of Forensic Pathology, School of Forensic Medicine, Southern Medical University, Guangzhou, China
| | - Qi Wang
- Department of Forensic Pathology, School of Forensic Medicine, Southern Medical University, Guangzhou, China.
| | - Hui-Jun Wang
- Department of Forensic Pathology, School of Forensic Medicine, Southern Medical University, Guangzhou, China.
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Connors E, Soto-Dávila M, Hossain A, Vasquez I, Gnanagobal H, Santander J. Identification and validation of reliable Aeromonas salmonicida subspecies salmonicida reference genes for differential gene expression analyses. INFECTION GENETICS AND EVOLUTION 2019; 73:314-321. [DOI: 10.1016/j.meegid.2019.05.011] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/14/2019] [Revised: 05/09/2019] [Accepted: 05/14/2019] [Indexed: 01/19/2023]
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Ono CT, Yu Z, Kikuchi Y, Kunii Y, Hino M, Matsumoto J, Nagaoka A, Ito J, Iwasaki Y, Hagihara H, Miyakawa T, Yoshida M, Saito Y, Niwa SI, Yabe H, Kakita A, Tomita H. Minimal amount of tissue-based pH measurement to improve quality control in neuropsychiatric post-mortem brain studies. Psychiatry Clin Neurosci 2019; 73:566-573. [PMID: 31102310 DOI: 10.1111/pcn.12863] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/25/2018] [Revised: 05/01/2019] [Accepted: 05/13/2019] [Indexed: 12/23/2022]
Abstract
AIM Tissue pH and RNA integrity are crucial quality-control indicators of human post-mortem brain tissues in the identification of the pathogeneses of neuropsychiatric disorders, but pH has not been measured as often due to limitations in the amount of tissue available. This study was designed to develop and validate a protocol for tissue pH evaluation using a minimal amount of human post-mortem tissues. METHODS A procedure that included a proper ratio of brain tissue weight to water for homogenization and the duration of homogenization was designed based on preliminary experiments using mouse brain tissues. The minimal (10 mg) and typical (100 mg) amounts of post-mortem brain tissue from 52 subjects were homogenized in 5 volumes (50 μL/10 mg tissue) and 10 volumes (1000 μL/100 mg tissue) of nuclease-free water and subjected to pH measurements using an InLab Ultra micro pH electrode. RESULTS The pH values based on the new protocol using a minimal amount of tissue significantly correlated with measurements of the standard protocol (r2 = 0.86). The correlation coefficients of the pH values between gray and white matter of the same brain region, and the values between different brain regions were 0.73 and 0.54, respectively. CONCLUSION The proposed protocol used one-tenth of the tissue amount of current standard protocol and enabled us to evaluate the exact quality of post-mortem brain tissue subjected to subsequent analyses. The application of this protocol may improve the detection of biological phenomena of interest in post-mortem brain studies by diminishing confounding factors.
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Affiliation(s)
- Chiaki T Ono
- Department of Disaster Psychiatry, International Research Institute of Disaster Psychiatry, Tohoku University, Sendai, Japan
| | - Zhiqian Yu
- Department of Disaster Psychiatry, International Research Institute of Disaster Psychiatry, Tohoku University, Sendai, Japan
| | - Yoshie Kikuchi
- Department of Disaster Psychiatry, International Research Institute of Disaster Psychiatry, Tohoku University, Sendai, Japan
| | - Yasuto Kunii
- Department of Neuropsychiatry, School of Medicine, Fukushima Medical University, Fukushima, Japan.,Department of Psychiatry, Aizu Medical Center, Fukushima Medical University, Fukushima, Japan
| | - Mizuki Hino
- Department of Neuropsychiatry, School of Medicine, Fukushima Medical University, Fukushima, Japan
| | - Junya Matsumoto
- Department of Neuropsychiatry, School of Medicine, Fukushima Medical University, Fukushima, Japan
| | - Atsuko Nagaoka
- Department of Neuropsychiatry, School of Medicine, Fukushima Medical University, Fukushima, Japan
| | - Junko Ito
- Department of Pathology, Brain Research Institute, Niigata University, Niigata, Japan
| | - Yasushi Iwasaki
- Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Nagakute, Japan
| | - Hideo Hagihara
- Division of Systems Medical Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Japan
| | - Tsuyoshi Miyakawa
- Division of Systems Medical Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Japan
| | - Mari Yoshida
- Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Nagakute, Japan
| | - Yuko Saito
- Department of Pathology and Laboratory Medicine, National Center Hospital, National Center of Neurology and Psychiatry, Kodaira, Japan
| | - Shin-Ichi Niwa
- Department of Psychiatry, Aizu Medical Center, Fukushima Medical University, Fukushima, Japan
| | - Hirooki Yabe
- Department of Neuropsychiatry, School of Medicine, Fukushima Medical University, Fukushima, Japan
| | - Akiyoshi Kakita
- Department of Pathology, Brain Research Institute, Niigata University, Niigata, Japan
| | - Hiroaki Tomita
- Department of Disaster Psychiatry, International Research Institute of Disaster Psychiatry, Tohoku University, Sendai, Japan
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Gómez-Carballa A, Cebey-López M, Pardo-Seco J, Barral-Arca R, Rivero-Calle I, Pischedda S, Currás-Tuala MJ, Gómez-Rial J, Barros F, Martinón-Torres F, Salas A. A qPCR expression assay of IFI44L gene differentiates viral from bacterial infections in febrile children. Sci Rep 2019; 9:11780. [PMID: 31409879 PMCID: PMC6692396 DOI: 10.1038/s41598-019-48162-9] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2019] [Accepted: 07/16/2019] [Indexed: 11/29/2022] Open
Abstract
The diagnosis of bacterial infections in hospital settings is currently performed using bacterial culture from sterile site, but they are lengthy and limited. Transcriptomic biomarkers are becoming promising tools for diagnosis with potential applicability in clinical settings. We evaluated a RT-qPCR assay for a 2-transcript host expression signature (FAM89A and IFI44L genes) inferred from microarray data that allow to differentiate between viral and bacterial infection in febrile children. This assay was able to discriminate viral from bacterial infections (P-value = 1.04 × 10-4; AUC = 92.2%; sensitivity = 90.9%; specificity = 85.7%) and showed very high reproducibility regardless of the reference gene(s) used to normalize the data. Unexpectedly, the monogenic IFI44L expression signature yielded better results than those obtained from the 2-transcript test (P-value = 3.59 × 10-5; AUC = 94.1%; sensitivity = 90.9%; specificity = 92.8%). We validated this IFI44L signature in previously published microarray and whole-transcriptome data from patients affected by different types of viral and bacterial infections, confirming that this gene alone differentiates between both groups, thus saving time, effort, and costs. Herein, we demonstrate that host expression microarray data can be successfully translated into a fast, highly accurate and relatively inexpensive in vitro assay that could be implemented in the clinical routine.
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Affiliation(s)
- Alberto Gómez-Carballa
- Genetics, Vaccines and Infections Research Group (GENVIP), Instituto de Investigación Sanitaria de Santiago, Santiago de Compostela, Spain.
- Translational Pediatrics and Infectious Diseases, Department of Pediatrics, Hospital Clínico Universitario de Santiago de Compostela, Santiago de Compostela, Spain.
- Unidade de Xenética, Instituto de Ciencias Forenses, Facultade de Medicina, Universidade de Santiago de Compostela, and GenPoB Research Group, Instituto de Investigaciones Sanitarias (IDIS), Hospital Clínico Universitario de Santiago (SERGAS), Galicia, Spain.
| | - Miriam Cebey-López
- Genetics, Vaccines and Infections Research Group (GENVIP), Instituto de Investigación Sanitaria de Santiago, Santiago de Compostela, Spain
- Translational Pediatrics and Infectious Diseases, Department of Pediatrics, Hospital Clínico Universitario de Santiago de Compostela, Santiago de Compostela, Spain
| | - Jacobo Pardo-Seco
- Genetics, Vaccines and Infections Research Group (GENVIP), Instituto de Investigación Sanitaria de Santiago, Santiago de Compostela, Spain
- Translational Pediatrics and Infectious Diseases, Department of Pediatrics, Hospital Clínico Universitario de Santiago de Compostela, Santiago de Compostela, Spain
- Unidade de Xenética, Instituto de Ciencias Forenses, Facultade de Medicina, Universidade de Santiago de Compostela, and GenPoB Research Group, Instituto de Investigaciones Sanitarias (IDIS), Hospital Clínico Universitario de Santiago (SERGAS), Galicia, Spain
| | - Ruth Barral-Arca
- Genetics, Vaccines and Infections Research Group (GENVIP), Instituto de Investigación Sanitaria de Santiago, Santiago de Compostela, Spain
- Translational Pediatrics and Infectious Diseases, Department of Pediatrics, Hospital Clínico Universitario de Santiago de Compostela, Santiago de Compostela, Spain
| | - Irene Rivero-Calle
- Genetics, Vaccines and Infections Research Group (GENVIP), Instituto de Investigación Sanitaria de Santiago, Santiago de Compostela, Spain
- Translational Pediatrics and Infectious Diseases, Department of Pediatrics, Hospital Clínico Universitario de Santiago de Compostela, Santiago de Compostela, Spain
| | - Sara Pischedda
- Genetics, Vaccines and Infections Research Group (GENVIP), Instituto de Investigación Sanitaria de Santiago, Santiago de Compostela, Spain
- Translational Pediatrics and Infectious Diseases, Department of Pediatrics, Hospital Clínico Universitario de Santiago de Compostela, Santiago de Compostela, Spain
| | - María José Currás-Tuala
- Genetics, Vaccines and Infections Research Group (GENVIP), Instituto de Investigación Sanitaria de Santiago, Santiago de Compostela, Spain
- Translational Pediatrics and Infectious Diseases, Department of Pediatrics, Hospital Clínico Universitario de Santiago de Compostela, Santiago de Compostela, Spain
| | - José Gómez-Rial
- Genetics, Vaccines and Infections Research Group (GENVIP), Instituto de Investigación Sanitaria de Santiago, Santiago de Compostela, Spain
- Translational Pediatrics and Infectious Diseases, Department of Pediatrics, Hospital Clínico Universitario de Santiago de Compostela, Santiago de Compostela, Spain
| | - Francisco Barros
- Unidad de Medicina Molecular, Fundación Pública Galega de Medicina Xenómica, CIBERER, Santiago de Compostela, Spain
| | - Federico Martinón-Torres
- Genetics, Vaccines and Infections Research Group (GENVIP), Instituto de Investigación Sanitaria de Santiago, Santiago de Compostela, Spain
- Translational Pediatrics and Infectious Diseases, Department of Pediatrics, Hospital Clínico Universitario de Santiago de Compostela, Santiago de Compostela, Spain
| | - Antonio Salas
- Genetics, Vaccines and Infections Research Group (GENVIP), Instituto de Investigación Sanitaria de Santiago, Santiago de Compostela, Spain
- Translational Pediatrics and Infectious Diseases, Department of Pediatrics, Hospital Clínico Universitario de Santiago de Compostela, Santiago de Compostela, Spain
- Unidade de Xenética, Instituto de Ciencias Forenses, Facultade de Medicina, Universidade de Santiago de Compostela, and GenPoB Research Group, Instituto de Investigaciones Sanitarias (IDIS), Hospital Clínico Universitario de Santiago (SERGAS), Galicia, Spain
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Jovčevska I, Zottel A, Šamec N, Mlakar J, Sorokin M, Nikitin D, Buzdin AA, Komel R. High FREM2 Gene and Protein Expression Are Associated with Favorable Prognosis of IDH-WT Glioblastomas. Cancers (Basel) 2019; 11:cancers11081060. [PMID: 31357584 PMCID: PMC6721429 DOI: 10.3390/cancers11081060] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2019] [Revised: 07/24/2019] [Accepted: 07/25/2019] [Indexed: 11/16/2022] Open
Abstract
World Health Organization grade IV diffuse gliomas, known as glioblastomas, are the most common malignant brain tumors, and they show poor prognosis. Multimodal treatment of surgery followed by radiation and chemotherapy is not sufficient to increase patient survival, which is 12 to 18 months after diagnosis. Despite extensive research, patient life expectancy has not significantly improved over the last decade. Previously, we identified FREM2 and SPRY1 as genes with differential expression in glioblastoma cell lines compared to nonmalignant astrocytes. In addition, the FREM2 and SPRY1 proteins show specific localization on the surface of glioblastoma cells. In this study, we explored the roles of the FREM2 and SPRY1 genes and their proteins in glioblastoma pathology using human tissue samples. We used proteomic, transcriptomic, and bioinformatics approaches to detect changes at different molecular levels. We demonstrate increased FREM2 protein expression levels in glioblastomas compared to reference samples. At the transcriptomic level, both FREM2 and SPRY1 show increased expression in tissue samples of different glioma grades compared to nonmalignant brain tissue. To broaden our experimental findings, we analyzed The Cancer Genome Atlas glioblastoma patient datasets. We discovered higher FREM2 and SPRY1 gene expression levels in glioblastomas compared to lower grade gliomas and reference samples. In addition, we observed that low FREM2 expression was associated with progression of IDH-mutant low-grade glioma patients. Multivariate analysis showed positive association between FREM2 and favorable prognosis of IDH-wild type glioblastoma. We conclude that FREM2 has an important role in malignant progression of glioblastoma, and we suggest deeper analysis to determine its involvement in glioblastoma pathology.
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Affiliation(s)
- Ivana Jovčevska
- Medical Centre for Molecular Biology, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia.
| | - Alja Zottel
- Medical Centre for Molecular Biology, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia
| | - Neja Šamec
- Medical Centre for Molecular Biology, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia
| | - Jernej Mlakar
- Institute of Pathology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia
| | - Maxim Sorokin
- Laboratory of Clinical and Genomic Bioinformatics, I. M. Sechenov First Moscow State Medical University, 119146 Moscow, Russia
- Omicsway Corp., Walnut, CA 91789, USA
- Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia
| | - Daniil Nikitin
- Laboratory of Clinical and Genomic Bioinformatics, I. M. Sechenov First Moscow State Medical University, 119146 Moscow, Russia
- Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia
| | - Anton A Buzdin
- Laboratory of Clinical and Genomic Bioinformatics, I. M. Sechenov First Moscow State Medical University, 119146 Moscow, Russia
- Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia
- Oncobox Ltd., 121205 Moscow, Russia
| | - Radovan Komel
- Medical Centre for Molecular Biology, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia
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APOBEC3B Gene Expression in Ductal Carcinoma In Situ and Synchronous Invasive Breast Cancer. Cancers (Basel) 2019; 11:cancers11081062. [PMID: 31357602 PMCID: PMC6721358 DOI: 10.3390/cancers11081062] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2019] [Revised: 07/17/2019] [Accepted: 07/25/2019] [Indexed: 11/17/2022] Open
Abstract
The underlying mechanism of the progression of ductal carcinoma in situ (DCIS), a non-obligate precursor of invasive breast cancer (IBC), has yet to be elucidated. In IBC, Apolipoprotein B mRNA Editing Enzyme, Catalytic Polypeptide-Like 3B (APOBEC3B) is upregulated in a substantial proportion of cases and is associated with higher mutational load and poor prognosis. However, APOBEC3B expression has never been studied in DCIS. We performed mRNA expression analysis of APOBEC3B in synchronous DCIS and IBC and surrounding normal cells. RNA was obtained from 53 patients. The tumors were categorized based on estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (Her2) and phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) mutation status. APOBEC3B mRNA levels were measured by RT-qPCR. The expression levels of paired DCIS and adjacent IBC were compared, including subgroup analyses. The normal cells expressed the lowest levels of APOBEC3B. No differences in expression were found between DCIS and IBC. Subgroup analysis showed that APOBEC3B was the highest in the ER subgroups of DCIS and IBC. While there was no difference in APOBEC3B between wild-type versus mutated PIK3CA DCIS, APOBEC3B was higher in wild-type versus PIK3CA-mutated IBC. In summary, our data show that APOBEC3B is already upregulated in DCIS. This suggests that APOBEC3B could already play a role in early carcinogenesis. Since APOBEC3B is a gain-of-function mutagenic enzyme, patients could benefit from the therapeutic targeting of APOBEC3B in the early non-invasive stage of breast cancer.
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Tani N, Ikeda T, Aoki Y, Shida A, Oritani S, Ishikawa T. Evaluation of screening for drug use using postmortem prolactin levels in serum and cerebrospinal fluid. Hum Exp Toxicol 2019; 38:1244-1253. [PMID: 31319705 DOI: 10.1177/0960327119864139] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Prolactin (PRL) levels can usually be controlled by PRL-inhibiting psychiatric drugs that include anti-dopamine agents. However, the use of dopamine (DA) antagonists may lead to hyperprolactinemia under certain clinical conditions. The aim of this study was to investigate postmortem PRL levels as potential markers of drug abuse, especially that of DA antagonists, in autopsy cases. We examined 121 autopsy cases, excluding cases involving acute hypoxia/ischemia, such as asphyxia, because PRL concentrations are reportedly increased under acute hypoxic conditions. Detected drugs were classified as either DA antagonists, stimulants, psychotropic drugs other than DA antagonists, or other non-psychotropic drugs, and many cases had no detected drugs. Samples comprised blood collected from the right heart chamber and cerebrospinal fluid (CSF). PRL protein level was measured by chemiluminescent immunoassay, and PRL gene expression in the anterior pituitary of autopsy cases was analyzed by reverse transcription-polymerase chain reaction. The PRL-positive cell ratio in the anterior pituitary gland was also measured by immunohistochemical analysis. The results indicated that PRL levels in the serum and CSF were higher in DA antagonist cases than in other cases. PRL levels in the serum and CSF also correlated with the PRL gene expression in cases with abuse of DA antagonists. However, no significant difference in the PRL-positive cell ratio in the anterior pituitary gland was evident between any of the classes of drug-detected and drug-undetected cases. These results suggest that postmortem measurements of PRL transcription levels may be useful for diagnosing cases of DA antagonist use.
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Affiliation(s)
- N Tani
- Department of Legal Medicine, Osaka City University Medical School, Osaka, Japan.,Forensic Autopsy Section, Medico-legal Consultation and Postmortem Investigation Support Center, c/o Department of Legal Medicine, Osaka City University Medical School, Osaka, Japan
| | - T Ikeda
- Department of Legal Medicine, Osaka City University Medical School, Osaka, Japan.,Forensic Autopsy Section, Medico-legal Consultation and Postmortem Investigation Support Center, c/o Department of Legal Medicine, Osaka City University Medical School, Osaka, Japan
| | - Y Aoki
- Department of Legal Medicine, Osaka City University Medical School, Osaka, Japan
| | - A Shida
- Department of Legal Medicine, Osaka City University Medical School, Osaka, Japan
| | - S Oritani
- Department of Legal Medicine, Osaka City University Medical School, Osaka, Japan
| | - T Ishikawa
- Department of Legal Medicine, Osaka City University Medical School, Osaka, Japan.,Forensic Autopsy Section, Medico-legal Consultation and Postmortem Investigation Support Center, c/o Department of Legal Medicine, Osaka City University Medical School, Osaka, Japan
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43
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Tani N, Ikeda T, Aoki Y, Shida A, Oritani S, Ishikawa T. Pathophysiological significance of clock genes BMAL1 and PER2 as erythropoietin-controlling factors in acute blood hemorrhage. Hum Cell 2019; 32:275-284. [PMID: 30941700 DOI: 10.1007/s13577-019-00248-2] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2018] [Accepted: 03/13/2019] [Indexed: 12/18/2022]
Abstract
This study aimed to characterize the pathophysiology, including possible correlations, of clock gene expression and erythropoietin (EPO) production in the acute stage of blood hemorrhage. Specimens of human cortical tissues (right and left kidneys) and cardiac blood were collected at autopsy from 52 cases following mortality due to acute-stage blood hemorrhage following sharp instrument injury. BMAL1 and PER2 mRNA levels were determined by reverse transcription-polymerase chain reaction; BMAL1 and PER2 protein levels were assessed using immunohistochemistry; BMAL1 protein levels were quantitatively measured by western blotting; and serum EPO levels were measured by chemiluminescent enzyme immunoassay. Separately, a rat model of hemorrhagic conditions was generated and used to confirm the results obtained with autopsy-derived specimens. A positive correlation was observed between BMAL1 protein and serum EPO levels, but not between BMAL1 mRNA levels and serum EPO levels. We also noted that Per2 mRNA expression became elevated in humans who survived for > 3 h after acute hemorrhagic events, with subsequent decreases in serum EPO levels. The rat model showed that even short (30-min) intervals of blood loss yielded increases in both Bmal1 mRNA and serum EPO levels; longer (60-min) intervals resulted in increases in Per2 mRNA expression along with decreases in serum EPO. Thus, the acute-stage human hemorrhage cases and the rat hemorrhage model yielded similar tendencies for clock gene expression and EPO secretion. In conclusion, our results indicated that clock genes are involved in the regulation of EPO production during the early stages of hypoxia/ischemia resulting from the acute hemorrhagic events.
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Affiliation(s)
- Naoto Tani
- Department of Legal Medicine, Osaka City University Medical School, Asahi-Machi 1-4-3, Abeno, Osaka, 545-8585, Japan.
- Forensic Autopsy Section, Medico-Legal Consultation and Postmortem Investigation Support Center (MLCPI-SC), Osaka, Japan.
| | - Tomoya Ikeda
- Department of Legal Medicine, Osaka City University Medical School, Asahi-Machi 1-4-3, Abeno, Osaka, 545-8585, Japan
- Forensic Autopsy Section, Medico-Legal Consultation and Postmortem Investigation Support Center (MLCPI-SC), Osaka, Japan
| | - Yayoi Aoki
- Department of Legal Medicine, Osaka City University Medical School, Asahi-Machi 1-4-3, Abeno, Osaka, 545-8585, Japan
| | - Alissa Shida
- Department of Legal Medicine, Osaka City University Medical School, Asahi-Machi 1-4-3, Abeno, Osaka, 545-8585, Japan
| | - Shigeki Oritani
- Department of Legal Medicine, Osaka City University Medical School, Asahi-Machi 1-4-3, Abeno, Osaka, 545-8585, Japan
| | - Takaki Ishikawa
- Department of Legal Medicine, Osaka City University Medical School, Asahi-Machi 1-4-3, Abeno, Osaka, 545-8585, Japan
- Forensic Autopsy Section, Medico-Legal Consultation and Postmortem Investigation Support Center (MLCPI-SC), Osaka, Japan
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Kaur R, Gupta M, Singh S, Pandher S. Evaluation and validation of experimental condition-specific reference genes for normalization of gene expression in Asia II-I Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). Gene Expr Patterns 2019; 34:119058. [PMID: 31185291 DOI: 10.1016/j.gep.2019.119058] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2019] [Revised: 05/31/2019] [Accepted: 05/31/2019] [Indexed: 01/08/2023]
Abstract
Functional genomics in whitefly, Bemisia tabaci is gaining impetus due to its polyphagous nature, worldwide distribution and recently sequenced whole genome. These studies require an in-depth evaluation and validation of reference genes in different development stages and variable experimental setups. Normalization with reference genes is an essential step in the gene expression studies. Rather than selecting a reference gene empirically, the suitability of these genes must be validated for an individual organism, its specific stage or even for particular experimental conditions. The Quantitative real-time polymerase chain reaction (RT-qPCR) has evolved as an efficient and widely used technique for precise monitoring of gene expression. The prime focus of this study was to identify candidate reference genes in different developmental stages (adults, nymphs, eggs), sex (male and female), hosts (Gossypium hirsutum, G. arboreum), and under insecticidal and starvation stress. Expression stability of these genes in different experimental samples was evaluated by employing five different computational algorithms such as NormFinder, BestKeeper, Comparative delta-CT, geNorm and RefFinder. Our results identified a different set of reference genes under each experimental setup such as electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), ubiquitin (UBIQ) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as best reference genes in adult whitefly. In addition, glutathione S-transferase (GST) in eggs, cyclophilin (CYCLOPH) in red eyed nymph, GAPDH in third instar, tubulin (Tub) in female, ubiquitin ribosomal protein S27 (UBIRPS2) in male, succinate dehydrogenase complex subunit B (SDHB) under insecticidal stress, ETF-QO under starvation stress, UBIRPS2 under host influence were the top most stable genes. Our studies report the importance of selection of specific reference genes for accurate gene expression studies under various experimental setups in B. tabaci.
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Affiliation(s)
- Ramandeep Kaur
- Punjab Agricultural University, Regional Research Station, Faridkot, 151203, Punjab, India
| | - Mridula Gupta
- Punjab Agricultural University, Regional Research Station, Faridkot, 151203, Punjab, India
| | - Satnam Singh
- Punjab Agricultural University, Regional Research Station, Faridkot, 151203, Punjab, India.
| | - Suneet Pandher
- Punjab Agricultural University, Regional Research Station, Faridkot, 151203, Punjab, India
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Wang H, Ma J, Xu H, Lyu Y, Tao L, Li W, Zeng Y, Ma K, Xiao B, Chen L. Early postmortem interval (EPMI) estimation using differentially expressed gene transcripts. Leg Med (Tokyo) 2019; 38:83-91. [PMID: 31108272 DOI: 10.1016/j.legalmed.2019.04.008] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2019] [Revised: 04/07/2019] [Accepted: 04/30/2019] [Indexed: 10/26/2022]
Abstract
Genes differentially expressed after death were selected to construct a mathematical model for early postmortem interval estimation. Sprague Dawley rats were sacrificed and placed at temperatures of 4 °C, 15 °C, 25 °C, and 35 °C. Brain tissues were collected at 0, 6, 12, 18, and 24 h after death and total RNA was extracted. Changes in gene transcript levels after death were detected using microarray expression profiling and differentially expressed genes was screened. Expanded experiments were performed to validate gene transcript levels at different temperatures using the reverse transcription real-time quantitative polymerase chain reaction. Six genes with high coefficients of determination were chosen for construction of mathematical models. Optimal ternary cubic equations were built using R software with temperature, postmortem interval and ΔCq defined as the independent variable x, y and z, respectively. Equations were converted into a three-dimensional visual statistical model using MATLAB. Animal samples were used to validate the mathematical models. Results showed that the 5srRNA showed best stability at four temperatures. The genes Ninj2, Grifin, Arpp19, and Hopx showed high coefficients of determination (>80%) and low error (<3h) in verification experiments which indicate that they are potential markers for early postmortem interval estimation.
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Affiliation(s)
- Hui Wang
- Department of Forensic Medicine, School of Basic Medical Sciences, Fudan University, 131 Dongan Road, Shanghai 200032, PR China
| | - Jianlong Ma
- Shenzhen Institute of Criminal Science and Technology, Investigation Department of Shenzhen Public Security Bureau, Key Laboratory of Forensic Pathology, Ministry of Public Security, Shenzhen 518000, PR China
| | - Hongmei Xu
- Department of Forensic Medicine, School of Basic Medical Sciences, Fudan University, 131 Dongan Road, Shanghai 200032, PR China
| | - Yehui Lyu
- Shanghai University of Medicine & Health Sciences, 279 ZhouzhuHwy, Shanghai 201318, PR China
| | - Li Tao
- Department of Forensic Medicine, School of Basic Medical Sciences, Fudan University, 131 Dongan Road, Shanghai 200032, PR China
| | - Wencan Li
- Forensic Lab, Criminal Science and Technology Institute, Pudong Branch, Shanghai Public Security Bureau, 255 Yanzhong Road, Shanghai 200125, PR China
| | - Yan Zeng
- Children's Hospital, Fudan University, 399 Wanyuan Road, Shanghai 201102, PR China
| | - Kaijun Ma
- Forensic Lab, Criminal Science and Technology Institute, Shanghai Public Security Bureau, 803 North Zhongshan Road, Shanghai 200082, PR China
| | - Bi Xiao
- Forensic Lab, Criminal Science and Technology Institute, Shanghai Public Security Bureau, 803 North Zhongshan Road, Shanghai 200082, PR China.
| | - Long Chen
- Department of Forensic Medicine, School of Basic Medical Sciences, Fudan University, 131 Dongan Road, Shanghai 200032, PR China.
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46
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Buonpane C, Ares G, Benyamen B, Yuan C, Hunter CJ. Identification of suitable reference microRNA for qPCR analysis in pediatric inflammatory bowel disease. Physiol Genomics 2019; 51:169-175. [PMID: 30978148 DOI: 10.1152/physiolgenomics.00126.2018] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Pediatric inflammatory bowel disease (IBD) accounts for 10-15% of IBD and is associated with considerable morbidity for patients. Dysregulated microRNAs (miRNA, miR), small noncoding RNA molecules that modulate gene expression, have been the target of research in IBD diagnosis, surveillance, and therapy. Proper selection of reference genes, which are a prerequisite for accurate measurement of miRNA expression, is currently lacking. We hypothesize that appropriate normalization requires unique reference genes for different tissue and disease types. Through the study of 28 pediatric intestinal samples, we sought to create a protocol for selection of suitable endogenous reference genes. Candidate reference genes (miR-16, 193a, 27a, 103a, 191) were analyzed by RT-quantitative (q)PCR. Criteria used for designation of suitable reference genes were as follows: 1) ubiquitous: present in all tissue samples with quantification cycle value 15-35; 2) uniform expression: no differential expression between control and disease samples (P > 0.05); 3) stability: stability value <0.5 by NormFinder. Our results suggest the use of miR-27a/191 for Crohn's disease small bowel, none of the five candidate genes for Crohn's disease colon, and miR-16/27a for ulcerative colitis. Additionally, target miR-874 had differential expression when normalized with different reference genes. Our results demonstrate that reference gene choice for qPCR analysis has a significant effect on study results and that proper data normalization is imperative.
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Affiliation(s)
- Christie Buonpane
- Department of Pediatrics, Feinberg School of Medicine, Northwestern University , Chicago, Illinois.,Ann and Robert H. Lurie Children's Hospital of Chicago , Chicago, Illinois
| | - Guillermo Ares
- Ann and Robert H. Lurie Children's Hospital of Chicago , Chicago, Illinois.,Department of Surgery, University of Illinois at Chicago , Chicago, Illinois
| | - Beshoy Benyamen
- Department of Pediatrics, Feinberg School of Medicine, Northwestern University , Chicago, Illinois.,Ann and Robert H. Lurie Children's Hospital of Chicago , Chicago, Illinois
| | - Carrie Yuan
- Department of Pediatrics, Feinberg School of Medicine, Northwestern University , Chicago, Illinois
| | - Catherine J Hunter
- Department of Pediatrics, Feinberg School of Medicine, Northwestern University , Chicago, Illinois.,Ann and Robert H. Lurie Children's Hospital of Chicago , Chicago, Illinois
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Tu C, Du T, Ye X, Shao C, Xie J, Shen Y. Using miRNAs and circRNAs to estimate PMI in advanced stage. Leg Med (Tokyo) 2019; 38:51-57. [PMID: 30986695 DOI: 10.1016/j.legalmed.2019.04.002] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2018] [Revised: 03/26/2019] [Accepted: 04/01/2019] [Indexed: 12/13/2022]
Abstract
In our previous study, we evaluated the stability of multi-RNA markers in heart, liver and skeletal muscle tissues of mice within 8 days after death and concluded that microRNAs (miRNAs) and circular (circRNAs) were more stable as reference genes in dead bodies than other kinds of RNAs. Based on their tissue-specific expression, we obtained reference genes for three kinds of tissues: miR-122, miR-133a and 18S in heart tissues; LC-Ogdh, circ-AFF1 and miR-122 in liver tissues; and miR-133a, circ-AFF1 in skeletal muscle tissues. For the estimation of post mortem interval (PMI), we also selected suitable biomarkers, which exhibited the best correlation coefficient with PMI. In our stability analysis of multi-RNA markers, Gapdh, Rps18, U6 and β-actin were unstable and selected as candidate target biomarkers. By analyzing the correlation between the expression levels of candidate target biomarkers and PMI, we obtained suitable target biomarkers for the three kinds of tissues, respectively. Finally, we established mathematical models of PMI estimation using the above selected reference genes and target biomarkers. The low estimated error in the validated samples demonstrated that PMI in advanced stage could be accurately predicted by real-time quantitative polymerase chain reaction (qPCR) through systematically selected effective reference genes and target biomarkers. Besides, combining the estimated results of various tissues and multi-biomarkers could improve the accuracy of PMI estimation.
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Affiliation(s)
- Chunyan Tu
- Department of Forensic Medicine, School of Basic Medical Sciences, Fudan University, Shanghai 200032, PR China
| | - Tieshuai Du
- Department of Forensic Medicine, School of Basic Medical Sciences, Fudan University, Shanghai 200032, PR China
| | - Xing Ye
- Department of Forensic Medicine, School of Basic Medical Sciences, Fudan University, Shanghai 200032, PR China
| | - Chengchen Shao
- Department of Forensic Medicine, School of Basic Medical Sciences, Fudan University, Shanghai 200032, PR China
| | - Jianhui Xie
- Department of Forensic Medicine, School of Basic Medical Sciences, Fudan University, Shanghai 200032, PR China.
| | - Yiwen Shen
- Department of Forensic Medicine, School of Basic Medical Sciences, Fudan University, Shanghai 200032, PR China.
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Cappato S, Giacopelli F, Tonachini L, Ravazzolo R, Bocciardi R. Identification of reference genes for quantitative PCR during C3H10T1/2 chondrogenic differentiation. Mol Biol Rep 2019; 46:3477-3485. [PMID: 30847849 PMCID: PMC6548758 DOI: 10.1007/s11033-019-04713-x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2018] [Accepted: 02/20/2019] [Indexed: 01/03/2023]
Abstract
C3H10T1/2, a mouse mesenchymal stem cell line, is a well-known in vitro model of chondrogenesis that can be easily employed to recapitulate some of the mechanisms intervening in this process. Moreover, these cells can be used to validate the effect of candidate molecules identified by high throughput screening approaches applied to the development of targeted therapy for human disorders in which chondrogenic differentiation may be involved, as in conditions characterized by heterotopic endochondral bone formation. Chondrogenic differentiation of C3H10T1/2 cells can be monitored by applying quantitative polymerase chain reaction (qPCR), one of the most sensitive methods that allows detection of small dynamic changes in gene expression between samples obtained under different experimental conditions. In this work, we have used qPCR to monitor the expression of specific markers during chondrogenic differentiation of C3H10T1/2 cells in micromass cultures. Then we have applied the geNorm approach to identify the most stable reference genes suitable to get a robust normalization of the obtained expression data. Among 12 candidate reference genes (Ap3d1, Csnk2a2, Cdc40, Fbxw2, Fbxo38, Htatsf1, Mon2, Pak1ip1, Zfp91, 18S, ActB, GAPDH) we identified Mon2 and Ap3d1 as the most stable ones during chondrogenesis. ActB, GAPDH and 18S, the most commonly used in the literature, resulted to have an expression level too high compared to the differentiation markers (Sox9, Collagen type 2a1, Collagen type 10a1 and Collagen type 1a1), therefore are actually less recommended for these experimental conditions. In conclusion, we identified nine reference genes that can be equally used to obtain a robust normalization of the gene expression variation during the C3H10T1/2 chondrogenic differentiation.
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Affiliation(s)
- Serena Cappato
- Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health (DINOGMI), Università degli Studi di Genova, 16132, Genova, Italy
| | - Francesca Giacopelli
- Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health (DINOGMI), Università degli Studi di Genova, 16132, Genova, Italy
| | - Laura Tonachini
- Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health (DINOGMI), Università degli Studi di Genova, 16132, Genova, Italy
| | - Roberto Ravazzolo
- Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health (DINOGMI), Università degli Studi di Genova, 16132, Genova, Italy.,Medical Genetics Unit, IRCCS Istituto Giannina Gaslini, 16147, Genova, Italy
| | - Renata Bocciardi
- Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health (DINOGMI), Università degli Studi di Genova, 16132, Genova, Italy. .,Medical Genetics Unit, IRCCS Istituto Giannina Gaslini, 16147, Genova, Italy.
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Morphological and neurochemical changes in GABAergic neurons of the aging human inferior colliculus. Hear Res 2019; 377:318-329. [PMID: 30878270 DOI: 10.1016/j.heares.2019.02.005] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/04/2019] [Revised: 02/09/2019] [Accepted: 02/13/2019] [Indexed: 12/20/2022]
Abstract
It is well known that quality of hearing decreases with increasing age due to changes in the peripheral or central auditory pathway. Along with the decrease in the number of neurons the neurotransmitter profile is also affected in the various parts of the auditory system. Particularly, changes in the inhibitory neurons in the inferior colliculus (IC) are known to affect quality of hearing with aging. To date, there is no information about the status of the inhibitory neurotransmitter GABA in the human IC during aging. We have collected and processed inferior colliculi of persons aged 11-97 years at the time of death for morphometry and immunohistochemical expression of glutamic acid decarboxylase (GAD67) and parvalbumin. We used unbiased stereology to estimate the number of cresyl-violet and immunostained neurons. Quantitative real-time PCR was used to measure the relative expression of the GAD67 mRNA. We found that the number of total, GABAergic and PV-positive neurons significantly decreased with increasing age (p < 0.05). The proportion of GAD67-ir neurons to total number of neurons was also negatively associated with increasing age (p = 0.004), but there was no change observed in the proportion of PV-ir neurons relative to GABAergic neurons (p = 0.25). Further, the fold change in the levels of GAD67 mRNA was negatively correlated to age (p = 0.024). We conclude that the poorer quality of hearing with increasing age may be due to decreased expression of inhibitory neurotransmitters and the decline in the number of inhibitory neurons in the IC.
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Pombo MA, Ramos RN, Zheng Y, Fei Z, Martin GB, Rosli HG. Transcriptome-based identification and validation of reference genes for plant-bacteria interaction studies using Nicotiana benthamiana. Sci Rep 2019; 9:1632. [PMID: 30733563 PMCID: PMC6367355 DOI: 10.1038/s41598-018-38247-2] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2018] [Accepted: 12/20/2018] [Indexed: 12/17/2022] Open
Abstract
RT-qPCR is a widely used technique for the analysis of gene expression. Accurate estimation of transcript abundance relies strongly on a normalization that requires the use of reference genes that are stably expressed in the conditions analyzed. Initially, they were adopted from those used in Northern blot experiments, but an increasing number of publications highlight the need to find and validate alternative reference genes for the particular system under study. The development of high-throughput sequencing techniques has facilitated the identification of such stably expressed genes. Nicotiana benthamiana has been extensively used as a model in the plant research field. In spite of this, there is scarce information regarding suitable RT-qPCR reference genes for this species. Employing RNA-seq data previously generated from tomato plants, combined with newly generated data from N. benthamiana leaves infiltrated with Pseudomonas fluorescens, we identified and tested a set of 9 candidate reference genes. Using three different algorithms, we found that NbUbe35, NbNQO and NbErpA exhibit less variable gene expression in our pathosystem than previously used genes. Furthermore, the combined use of the first two is sufficient for robust gene expression analysis. We encourage employing these novel reference genes in future RT-qPCR experiments involving N. benthamiana and Pseudomonas spp.
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Affiliation(s)
- Marina A Pombo
- Instituto de Fisiología Vegetal, INFIVE, Universidad Nacional de La Plata, CONICET, La Plata, Buenos Aires, Argentina
| | - Romina N Ramos
- Instituto de Fisiología Vegetal, INFIVE, Universidad Nacional de La Plata, CONICET, La Plata, Buenos Aires, Argentina
| | - Yi Zheng
- Boyce Thompson Institute for Plant Research, 533 Tower Road, Ithaca, NY, 14853, USA
| | - Zhangjun Fei
- Boyce Thompson Institute for Plant Research, 533 Tower Road, Ithaca, NY, 14853, USA
- USDA-ARS Robert W. Holley Center for Agriculture and Health, Ithaca, NY, 14853, USA
| | - Gregory B Martin
- Boyce Thompson Institute for Plant Research, 533 Tower Road, Ithaca, NY, 14853, USA
- Section of Plant Pathology and Plant-Microbe Biology, School of Integrative Plant Science, Cornell University, Ithaca, NY, 14853, USA
| | - Hernan G Rosli
- Instituto de Fisiología Vegetal, INFIVE, Universidad Nacional de La Plata, CONICET, La Plata, Buenos Aires, Argentina.
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