|
1
|
Park C, Park J, Chang D, Kim S. Development of reference-based model for improved analysis of bacterial community. Food Res Int 2025; 211:116380. [PMID: 40356165 DOI: 10.1016/j.foodres.2025.116380] [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: 02/26/2025] [Revised: 03/24/2025] [Accepted: 04/15/2025] [Indexed: 05/15/2025]
Abstract
Probiotic bacteria play a vital role in maintaining gut microbial homeostasis and are widely used in various commercial products. Although 16S rRNA amplicon-based next-generation sequencing (NGS) is commonly used to analyze probiotic products, biases can arise from various 16S rRNA amplification regions, sequencing platforms, and library kits. In this study, a reference-based bias correction model was developed to correct sequencing biases. The model was validated using eight mock communities and 12 commercial products, which were analyzed across multiple NGS platforms and various 16S rRNA regions. Specific primer-probe assays were developed for accurate bacterial quantification, and their specificity was validated and used in conjunction with droplet digital PCR (ddPCR) to establish initial bacterial ratios within communities. Analysis of the mock communities revealed platform- and region-specific biases, with specific species consistently over- or under-represented. Similarly, commercial product analyses have shown biased outcomes owing to varying sequencing protocols. The correction model, based on PCR efficiencies from the reference communities, successfully corrected biased ratios across different amplification regions and platforms to achieve results that closely matched the proportions predicted by ddPCR. The model effectively corrected the biases arising from the different polymerases. Notably, partial references containing approximately 40 % of the species achieved correction results that were comparable to those of the complete references. This approach demonstrates the potential for improving microbiome analysis accuracy within predictable ranges, and could serve as a model for addressing sequencing bias in metagenomic research.
Collapse
Affiliation(s)
- Changwoo Park
- Biometrology Group, Korea Research Institute of Standards and Science, Daejeon 34113, Republic of Korea.
| | - Jinyoung Park
- Biometrology Group, Korea Research Institute of Standards and Science, Daejeon 34113, Republic of Korea; Department of Precision Measurement, University of Science & Technology, Daejeon 34113, Republic of Korea.
| | - Dongho Chang
- Microbiome Convergence Research Center, Korea Research Institute of Bioscience & Biotechnology, Daejeon 34141, Republic of Korea.
| | - Seil Kim
- Biometrology Group, Korea Research Institute of Standards and Science, Daejeon 34113, Republic of Korea; Department of Precision Measurement, University of Science & Technology, Daejeon 34113, Republic of Korea.
| |
Collapse
|
|
2
|
Kehl A, Klein R, Steiger K, Aupperle-Lellbach H. Stability of microRNAs in Canine Serum-A Prerequisite for Use as Biomarkers in Tumour Diagnostics. Vet Sci 2025; 12:390. [PMID: 40284892 PMCID: PMC12031383 DOI: 10.3390/vetsci12040390] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2025] [Revised: 04/10/2025] [Accepted: 04/17/2025] [Indexed: 04/29/2025] Open
Abstract
Since microRNAs are released into the bloodstream and miRNA profiles are supposed to differ between healthy and tumour patients, miRNAs seem to have potential as biomarkers. An essential prerequisite for biomarkers in a routine diagnostic setup is their stability in serum over time. In this study, serum samples from 10 healthy dogs were analysed at different time points and under various temperature conditions (after 24 and 48 h, at 4° or 20 °C) for the copy number of eight miRNAs (miR-20b, 21, 122, 126, 192, 214, 222, 494) using ddPCR. The miR-21 had the highest copy number, whereas miR-494 had the lowest copy number in canine blood samples. The values of each miRNA varied individually between the dogs, showing a 5 to 10-fold range. Stability differed between the miRNAs, with miR-192 having the best stability. The copy number of miR-20b, miR-126 and miR-214 decreased not significantly during 48 h storage time. In contrast, miR-21, miR-122 and miR-222 were stable for 24 h only but decreased significantly after 48 h. The (in)stability of individual canine miRNAs must be considered when transferring study results into veterinary routine diagnostics, as the transport and storage conditions are variable. As far as possible, standardisation of sampling, storage and quantification of miRNAs is needed.
Collapse
Affiliation(s)
- Alexandra Kehl
- Laboklin GmbH&Co. KG, 97688 Bad Kissingen, Germany; (R.K.); (H.A.-L.)
- School of Medicine, Institute of Pathology, Technical University of Munich, 81675 München, Germany;
| | - Ruth Klein
- Laboklin GmbH&Co. KG, 97688 Bad Kissingen, Germany; (R.K.); (H.A.-L.)
| | - Katja Steiger
- School of Medicine, Institute of Pathology, Technical University of Munich, 81675 München, Germany;
| | - Heike Aupperle-Lellbach
- Laboklin GmbH&Co. KG, 97688 Bad Kissingen, Germany; (R.K.); (H.A.-L.)
- School of Medicine, Institute of Pathology, Technical University of Munich, 81675 München, Germany;
| |
Collapse
|
|
3
|
Gao N, Li J, Yang F, Yu D, Huo Y, Liu X, Ji Z, Xing Y, Zhang X, Yuan P, Liu J, Yan J. Forensic Age Estimation From Blood Samples by Combining DNA Methylation and MicroRNA Markers Using Droplet Digital PCR. Electrophoresis 2025; 46:424-432. [PMID: 40099741 DOI: 10.1002/elps.8133] [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] [Indexed: 03/20/2025]
Abstract
Age estimation is important in criminal investigations and forensic practice, and extensive studies have focused on age determination based on DNA methylation (DNAm) and miRNA markers. Interestingly, it has been reported that combining different types of molecular omics data helps build more accurate predictive models. However, few studies have compared the application of combined DNAm and miRNA data to predict age in the same cohort. In this study, a novel multiplex droplet digital PCR (ddPCR) system that allows for the simultaneous detection of age-associated DNAm and miRNA markers, including KLF14, miR-106b-5p, and two reference genes (C-LESS-C1 and RNU6B), was developed. Next, we examined and calculated the methylation levels of KLF14 and relative expression levels of miR-106b-5p in 132 blood samples. The collected data were used to establish age prediction models. Finally, the optimal models were evaluated using bloodstain samples. The results revealed that the random forest (RF) model had a minimum mean absolute deviation (MAD) value of 3.51 years and a maximum R2 of 0.84 for the validation sets in the combined age prediction models. However, the MAD was 5.66 years and the absolute error ranged from 3.16 to 10.54 years for bloodstain samples. Larger sample sizes and validation datasets are required to confirm these results in future studies. Overall, a stable method for the detection of KLF14, miR-106b-5p, C-LESS-C1, and RNU6B by 4-plex ddPCR was successfully established, and our study suggests that combining DNAm and miRNA data can improve the accuracy of age prediction, which has potential applications in forensic science.
Collapse
Affiliation(s)
- Niu Gao
- School of Forensic Medicine, Shanxi Medical University, Jinzhong, Shanxi, China
- Shanxi Key Laboratory of Forensic Medicine, Jinzhong, Shanxi, China
| | - Junli Li
- School of Forensic Medicine, Shanxi Medical University, Jinzhong, Shanxi, China
- Shanxi Key Laboratory of Forensic Medicine, Jinzhong, Shanxi, China
| | - Fenglong Yang
- School of Forensic Medicine, Shanxi Medical University, Jinzhong, Shanxi, China
- Shanxi Key Laboratory of Forensic Medicine, Jinzhong, Shanxi, China
| | - Daijing Yu
- School of Forensic Medicine, Shanxi Medical University, Jinzhong, Shanxi, China
- Shanxi Key Laboratory of Forensic Medicine, Jinzhong, Shanxi, China
| | - Yumei Huo
- School of Forensic Medicine, Shanxi Medical University, Jinzhong, Shanxi, China
- Shanxi Key Laboratory of Forensic Medicine, Jinzhong, Shanxi, China
| | - Xiaonan Liu
- School of Forensic Medicine, Shanxi Medical University, Jinzhong, Shanxi, China
- Shanxi Key Laboratory of Forensic Medicine, Jinzhong, Shanxi, China
| | - Zhimin Ji
- School of Forensic Medicine, Shanxi Medical University, Jinzhong, Shanxi, China
- Shanxi Key Laboratory of Forensic Medicine, Jinzhong, Shanxi, China
| | - Yangfeng Xing
- School of Forensic Medicine, Shanxi Medical University, Jinzhong, Shanxi, China
- Shanxi Key Laboratory of Forensic Medicine, Jinzhong, Shanxi, China
| | - Xiaomeng Zhang
- School of Forensic Medicine, Shanxi Medical University, Jinzhong, Shanxi, China
- Shanxi Key Laboratory of Forensic Medicine, Jinzhong, Shanxi, China
| | - Piao Yuan
- School of Forensic Medicine, Shanxi Medical University, Jinzhong, Shanxi, China
- Shanxi Key Laboratory of Forensic Medicine, Jinzhong, Shanxi, China
| | - Jinding Liu
- School of Forensic Medicine, Shanxi Medical University, Jinzhong, Shanxi, China
- Shanxi Key Laboratory of Forensic Medicine, Jinzhong, Shanxi, China
| | - Jiangwei Yan
- School of Forensic Medicine, Shanxi Medical University, Jinzhong, Shanxi, China
- Shanxi Key Laboratory of Forensic Medicine, Jinzhong, Shanxi, China
| |
Collapse
|
|
4
|
Dasgupta A, Tripathi A, Mitra A, Ghosh P, Santra MK, Mitra D. Human microRNA miR-197-3p positively regulates HIV-1 virion infectivity through its target DDX52 by stabilizing Vif protein expression. J Biol Chem 2025; 301:108198. [PMID: 39826696 PMCID: PMC11867528 DOI: 10.1016/j.jbc.2025.108198] [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: 06/10/2024] [Revised: 01/04/2025] [Accepted: 01/12/2025] [Indexed: 01/22/2025] Open
Abstract
MicroRNAs are a part of the integral regulatory mechanisms found in eukaryotic cells that help in maintaining cellular homeostasis by modulating the expression of target genes. However, during stress conditions like viral infection, the expression profile of the microRNAs change, thereby directly modulating the expression of viral genes and/or indirectly targeting the virus by regulating the host genes. The present study intends to identify previously uncharacterized cellular microRNAs, which are significantly modulated upon HIV-1 infection. With the available microarray data of five independent studies in the NCBI GEO database, 10 common yet functionally uncharacterized microRNAs that are deregulated during HIV-1 infection in humans were identified. Their expression profiles were validated in HIV-1 infected human peripheral blood mononuclear cells and a CD4+T cell line. Among them, miR-197-3p showed significant upregulation during HIV-1 infection in all the cell types tested and was selected for further characterization. We then found that miR-197-3p increases progeny virion infectivity through restricting the expression of DDX52. Interestingly, DDX52 showed a negative impact on virion infectivity by downregulating the HIV-1 viral infectivity factor (Vif) at the protein level. Mechanistically, our study also revealed that Vif, DDX52, and APOBEC3G form a complex, which might be responsible for Vif downregulation by proteasomal degradation. Taken together, our results demonstrate that miR-197-3p is a positive regulator of HIV-1 infectivity as it enhances the progeny virion infectivity by targeting DDX52, which is a negative regulator of Vif.
Collapse
Affiliation(s)
- Anindita Dasgupta
- National Centre for Cell Science, SP Pune University Campus, Pune, Maharashtra, India
| | - Anjali Tripathi
- National Centre for Cell Science, SP Pune University Campus, Pune, Maharashtra, India
| | - Alapani Mitra
- National Centre for Cell Science, SP Pune University Campus, Pune, Maharashtra, India
| | - Payel Ghosh
- Bioinformatics Centre, SP Pune University, Pune, Maharashtra, India
| | - Manas Kumar Santra
- National Centre for Cell Science, SP Pune University Campus, Pune, Maharashtra, India
| | - Debashis Mitra
- National Centre for Cell Science, SP Pune University Campus, Pune, Maharashtra, India.
| |
Collapse
|
|
5
|
Gleerup D, Trypsteen W, Fraley SI, De Spiegelaere W. Digital PCR in Virology: Current Applications and Future Perspectives. Mol Diagn Ther 2025; 29:43-54. [PMID: 39487879 DOI: 10.1007/s40291-024-00751-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/15/2024] [Indexed: 11/04/2024]
Abstract
Digital PCR (dPCR) has been used in the field of virology since its inception. Technological innovations in microfluidics more than a decade ago caused a sharp increase in its use. There is an emerging consensus that dPCR now outperforms quantitative PCR (qPCR) in the basic parameters such as precision, sensitivity, accuracy, repeatability and resistance to inhibitors. These strengths have led to several current applications in quantification, mutation detection and environmental DNA and RNA samples. In high throughput scenarios, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, the cost and throughput still significantly hampered the adaption of dPCR. There is much unexplored potential within the multiplexing capabilities of dPCR. This will allow simultaneous multi-target quantification and can also partially alleviate the throughput and cost drawback. In this review, we discuss the strengths and weaknesses of dPCR with a focus on virology applications and we discuss future applications. Finally, we discuss recent evolutions of the technology in the form of real-time dPCR and digital high-resolution melting.
Collapse
Affiliation(s)
- David Gleerup
- Laboratory of Veterinary Morphology, Faculty of Veterinary Medicine, Ghent University, Campus Merelbeke, Salisburylaan 133, 9820, Merelbeke, Belgium
- Ghent University Digital PCR Consortium, Ghent University, Ghent, Belgium
| | - Wim Trypsteen
- Ghent University Digital PCR Consortium, Ghent University, Ghent, Belgium
- Department of Internal Medicine and Pediatrics, HIV Cure Research Center, Ghent University and Ghent University Hospital, Ghent, Belgium
| | - Stephanie I Fraley
- Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
| | - Ward De Spiegelaere
- Laboratory of Veterinary Morphology, Faculty of Veterinary Medicine, Ghent University, Campus Merelbeke, Salisburylaan 133, 9820, Merelbeke, Belgium.
- Ghent University Digital PCR Consortium, Ghent University, Ghent, Belgium.
| |
Collapse
|
|
6
|
Lee SE, Koh UN, Fakhr R, Lim SK. Development of two step reverse transcription droplet digital PCR (RT-ddPCR) for simultaneous identification of saliva and semen. Leg Med (Tokyo) 2024; 71:102536. [PMID: 39490201 DOI: 10.1016/j.legalmed.2024.102536] [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: 08/16/2024] [Revised: 09/28/2024] [Accepted: 10/08/2024] [Indexed: 11/05/2024]
Abstract
Determination of the type of body fluids is essential for crime scene reconstruction and for improving the reliability of expert testimonies. Messenger RNA (mRNA) analysis by reverse transcription quantitative PCR (RT-qPCR) has been used in forensic genetics, particularly for body fluid identification. It is a relative quantification method that compares the Ct values of target and reference gene. Thus, the method is unsuitable for determining exact copy numbers of the target gene. To address this limitation, this study performed body fluid-specific mRNA analysis using two-step reverse transcription droplet digital PCR (RT-ddPCR), which is capable of absolute quantification. We found that RT-ddPCR was accurate and sensitive enough to detect as little as 1.5 copies/μl of complementary DNA (cDNA), making it suitable for application using casework samples. It was also highly specific for body fluids, as non-specific amplification did not occur. In addition, saliva-semen mixtures with ratios ranging from 1:50 to 50:1 were successfully identified. When comparing the results of RT-qPCR and RT-ddPCR, some samples were difficult to interpret because of the high Ct values of RT-qPCR. However, when the same samples were analyzed using RT-ddPCR, saliva and semen were distinctly identified. Thus, RT-ddPCR is useful for mixed samples (e.g., in sexual assault cases) with low amounts of DNA, which often leads to ambiguous results when using RT-qPCR. Other body fluids (e.g., vaginal fluid and menstrual blood) can also be identified by including additional markers. This study demonstrates the potential of RT-ddPCR for applications in forensic science.
Collapse
Affiliation(s)
- So Eun Lee
- Department of Forensic Sciences, Sungkyunkwan University, Suwon, Republic of Korea
| | - Un Na Koh
- Department of Forensic Sciences, Sungkyunkwan University, Suwon, Republic of Korea
| | - Rita Fakhr
- Department of Forensic Sciences, Sungkyunkwan University, Suwon, Republic of Korea
| | - Si-Keun Lim
- Department of Forensic Sciences, Sungkyunkwan University, Suwon, Republic of Korea; ID-Cell Forensics Co., Sungkyunkwan University, Suwon, Republic of Korea; Convergence Bio Forensic Institute (CBFI), Biomedical Institute for Convergence at Sungkyunkwan University (BICS), Sungkyunkwan University, Suwon, Republic of Korea.
| |
Collapse
|
|
7
|
Wascher M, Klaus CJ, Alvarado C, Panescu J, Quam M, Dannemiller KC, Tien JH. A mechanistic modeling and estimation framework for environmental pathogen surveillance. Math Biosci 2024; 377:109257. [PMID: 39173943 DOI: 10.1016/j.mbs.2024.109257] [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: 03/11/2024] [Revised: 05/30/2024] [Accepted: 07/08/2024] [Indexed: 08/24/2024]
Abstract
Environmental pathogen surveillance is a promising disease surveillance modality that has been widely adopted for SARS-CoV-2 monitoring. The highly variable nature of environmental pathogen data is a challenge for integrating these data into public health response. One source of this variability is heterogeneous infection both within an individual over the course of infection as well as between individuals in their pathogen shedding over time. We present a mechanistic modeling and estimation framework for connecting environmental pathogen data to the number of infected individuals. Infected individuals are modeled as shedding pathogen into the environment via a Poisson process whose rate parameter λt varies over the course of their infection. These shedding curves λt are themselves random, allowing for variation between individuals. We show that this results in a Poisson process for environmental pathogen levels with rate parameter a function of the number of infected individuals, total shedding over the course of infection, and pathogen removal from the environment. Theoretical results include determination of identifiable parameters for the model from environmental pathogen data and simple, explicit formulas for the likelihood for particular choices of individual shedding curves. We give a two step Bayesian inference framework, where the first step corresponds to calibration from data where the number of infected individuals is known, followed by an estimation step from environmental surveillance data when the number of infected individuals is unknown. We apply this modeling and estimation framework to synthetic data, as well as to an empirical case study of SARS-CoV-2 in environmental dust collected from isolation rooms housing university students. Both the synthetic data and empirical case study indicate high inter-individual variation in shedding, leading to wide credible intervals for the number of infected individuals. We examine how uncertainty in estimates of the number of infected individuals from environmental pathogen levels scales with the true number of infected individuals and model misspecification. While credible intervals for the number of infected individuals are wide, our results suggest that distinguishing between no infection and small-to-moderate levels of infection (≈10 infected individuals) may be possible, and that it is broadly possible to differentiate between moderate (≈40) and high (≈200) numbers of infected individuals.
Collapse
Affiliation(s)
- Matthew Wascher
- Division of Epidemiology, College of Public Health, The Ohio State University, United States of America; Department of Mathematics, Applied Mathematics, and Statistics, Case Western Reserve University, United States of America
| | - Colin J Klaus
- Mathematical Biosciences Institute and College of Public Health, The Ohio State University, United States of America
| | - Chance Alvarado
- Division of Epidemiology, College of Public Health, The Ohio State University, United States of America
| | - Jenny Panescu
- Department of Civil, Environmental and Geodetic Engineering, Division of Environmental Health Sciences, and Sustainability Institute, The Ohio State University, United States of America
| | - Mikkel Quam
- Division of Epidemiology, College of Public Health, The Ohio State University, United States of America
| | - Karen C Dannemiller
- Department of Civil, Environmental and Geodetic Engineering, Division of Environmental Health Sciences, and Sustainability Institute, The Ohio State University, United States of America
| | - Joseph H Tien
- Department of Mathematics and Division of Epidemiology, The Ohio State University, United States of America.
| |
Collapse
|
|
8
|
Stella M, Russo GI, Leonardi R, Carcò D, Gattuso G, Falzone L, Ferrara C, Caponnetto A, Battaglia R, Libra M, Barbagallo D, Di Pietro C, Pernagallo S, Barbagallo C, Ragusa M. Extracellular RNAs from Whole Urine to Distinguish Prostate Cancer from Benign Prostatic Hyperplasia. Int J Mol Sci 2024; 25:10079. [PMID: 39337566 PMCID: PMC11432375 DOI: 10.3390/ijms251810079] [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: 08/09/2024] [Revised: 09/13/2024] [Accepted: 09/17/2024] [Indexed: 09/30/2024] Open
Abstract
RNAs, especially non-coding RNAs (ncRNAs), are crucial players in regulating cellular mechanisms due to their ability to interact with and regulate other molecules. Altered expression patterns of ncRNAs have been observed in prostate cancer (PCa), contributing to the disease's initiation, progression, and treatment response. This study aimed to evaluate the ability of a specific set of RNAs, including long ncRNAs (lncRNAs), microRNAs (miRNAs), and mRNAs, to discriminate between PCa and the non-neoplastic condition benign prostatic hyperplasia (BPH). After selecting by literature mining the most relevant RNAs differentially expressed in biofluids from PCa patients, we evaluated their discriminatory power in samples of unfiltered urine from 50 PCa and 50 BPH patients using both real-time PCR and droplet digital PCR (ddPCR). Additionally, we also optimized a protocol for urine sample manipulation and RNA extraction. This two-way validation study allowed us to establish that miRNAs (i.e., miR-27b-3p, miR-574-3p, miR-30a-5p, and miR-125b-5p) are more efficient biomarkers for PCa compared to long RNAs (mRNAs and lncRNAs) (e.g., PCA3, PCAT18, and KLK3), as their dysregulation was consistently reported in the whole urine of patients with PCa compared to those with BPH in a statistically significant manner regardless of the quantification methodology performed. Moreover, a significant increase in diagnostic performance was observed when molecular signatures composed of different miRNAs were considered. Hence, the abovementioned circulating ncRNAs represent excellent potential non-invasive biomarkers in urine capable of effectively distinguishing individuals with PCa from those with BPH, potentially reducing cancer overdiagnosis.
Collapse
Affiliation(s)
- Michele Stella
- Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics "G. Sichel", University of Catania, 95123 Catania, Italy
| | - Giorgio Ivan Russo
- Department of Urology, Polyclinic Hospital, University of Catania, 95123 Catania, Italy
| | - Rosario Leonardi
- Casa di Cura Musumeci GECAS, 95030 Gravina di Catania, Italy
- Department of Medicine and Surgery, University of Enna KORE, 94100 Enna, Italy
| | - Daniela Carcò
- Istituto Oncologico del Mediterraneo, 95029 Viagrande, Italy
| | - Giuseppe Gattuso
- Department of Biomedical and Biotechnological Sciences, Oncologic, Clinical and General Pathology Section, University of Catania, 95123 Catania, Italy
| | - Luca Falzone
- Department of Biomedical and Biotechnological Sciences, Oncologic, Clinical and General Pathology Section, University of Catania, 95123 Catania, Italy
| | - Carmen Ferrara
- Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics "G. Sichel", University of Catania, 95123 Catania, Italy
| | - Angela Caponnetto
- Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics "G. Sichel", University of Catania, 95123 Catania, Italy
| | - Rosalia Battaglia
- Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics "G. Sichel", University of Catania, 95123 Catania, Italy
| | - Massimo Libra
- Department of Biomedical and Biotechnological Sciences, Oncologic, Clinical and General Pathology Section, University of Catania, 95123 Catania, Italy
| | - Davide Barbagallo
- Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics "G. Sichel", University of Catania, 95123 Catania, Italy
| | - Cinzia Di Pietro
- Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics "G. Sichel", University of Catania, 95123 Catania, Italy
| | - Salvatore Pernagallo
- DESTINA Genomica S.L., Health Sciences Technology Park (PTS), Av. de la Innovación 1, Building Business Innovation Center (BIC), 18016 Granada, Spain
| | - Cristina Barbagallo
- Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics "G. Sichel", University of Catania, 95123 Catania, Italy
| | - Marco Ragusa
- Department of Biomedical and Biotechnological Sciences, Section of Biology and Genetics "G. Sichel", University of Catania, 95123 Catania, Italy
| |
Collapse
|
|
9
|
Tom WA, Chandel DS, Jiang C, Krzyzanowski G, Fernando N, Olou A, Fernando MR. Genotype Characterization and MiRNA Expression Profiling in Usher Syndrome Cell Lines. Int J Mol Sci 2024; 25:9993. [PMID: 39337481 PMCID: PMC11432263 DOI: 10.3390/ijms25189993] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2024] [Revised: 09/10/2024] [Accepted: 09/14/2024] [Indexed: 09/30/2024] Open
Abstract
Usher syndrome (USH) is an inherited disorder characterized by sensorineural hearing loss (SNHL), retinitis pigmentosa (RP)-related vision loss, and vestibular dysfunction. USH presents itself as three distinct clinical types, 1, 2, and 3, with no biomarker for early detection. This study aimed to explore whether microRNA (miRNA) expression in USH cell lines is dysregulated compared to the miRNA expression pattern in a cell line derived from a healthy human subject. Lymphocytes from USH patients and healthy individuals were isolated and transformed into stable cell lines using Epstein-Barr virus (EBV). DNA from these cell lines was sequenced using a targeted panel to identify gene variants associated with USH types 1, 2, and 3. Microarray analysis was performed on RNA from both USH and control cell lines using NanoString miRNA microarray technology. Dysregulated miRNAs identified by the microarray were validated using droplet digital PCR technology. DNA sequencing revealed that two USH patients had USH type 1 with gene variants in USH1B (MYO7A) and USH1D (CDH23), while the other two patients were classified as USH type 2 (USH2A) and USH type 3 (CLRN-1), respectively. The NanoString miRNA microarray detected 92 differentially expressed miRNAs in USH cell lines compared to controls. Significantly altered miRNAs exhibited at least a twofold increase or decrease with a p value below 0.05. Among these miRNAs, 20 were specific to USH1, 14 to USH2, and 5 to USH3. Three miRNAs that are known as miRNA-183 family which are crucial for inner ear and retina development, have been significantly downregulated as compared to control cells. Subsequently, droplet digital PCR assays confirmed the dysregulation of the 12 most prominent miRNAs in USH cell lines. This study identifies several miRNA signatures in USH cell lines which may have potential utility in Usher syndrome identification.
Collapse
Affiliation(s)
- Wesley A Tom
- Molecular Diagnostic Research Laboratory, Center for Sensory Neuroscience, Boys Town National Research Hospital, Omaha, NE 68010, USA
| | - Dinesh S Chandel
- Molecular Diagnostic Research Laboratory, Center for Sensory Neuroscience, Boys Town National Research Hospital, Omaha, NE 68010, USA
| | - Chao Jiang
- Molecular Diagnostic Research Laboratory, Center for Sensory Neuroscience, Boys Town National Research Hospital, Omaha, NE 68010, USA
| | - Gary Krzyzanowski
- Molecular Diagnostic Research Laboratory, Center for Sensory Neuroscience, Boys Town National Research Hospital, Omaha, NE 68010, USA
| | - Nirmalee Fernando
- Molecular Diagnostic Research Laboratory, Center for Sensory Neuroscience, Boys Town National Research Hospital, Omaha, NE 68010, USA
| | - Appolinaire Olou
- Molecular Diagnostic Research Laboratory, Center for Sensory Neuroscience, Boys Town National Research Hospital, Omaha, NE 68010, USA
| | - M Rohan Fernando
- Molecular Diagnostic Research Laboratory, Center for Sensory Neuroscience, Boys Town National Research Hospital, Omaha, NE 68010, USA
| |
Collapse
|
|
10
|
Dass M, Ghai M. Development of a multiplex PCR assay and quantification of microbial markers by ddPCR for identification of saliva and vaginal fluid. Forensic Sci Int 2024; 362:112147. [PMID: 39067179 DOI: 10.1016/j.forsciint.2024.112147] [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: 04/12/2024] [Revised: 06/11/2024] [Accepted: 07/10/2024] [Indexed: 07/30/2024]
Abstract
The identification of biological fluids at crime scenes contributes to crime scene reconstruction and provides investigative leads. Traditional methods for body fluid identification are limited in terms of sensitivity and are mostly presumptive. Emerging methods based on mRNA and DNA methylation require high quality template source. An exploitable characteristic of body fluids is their distinct microbial profiles allowing for the discrimination of body fluids based on microbiome content. Microbial DNA is highly abundant within the body, robust and stable and can persist in the environment long after human DNA has degraded. 16S rRNA sequencing is the gold standard for microbial analysis; however, NGS is costly, and requires intricate workflows and interpretation. Also, species level resolution is not always achievable. Based on the current challenges, the first objective of this study was to develop a multiplex conventional PCR assay to identify vaginal fluid and saliva by targeting species-specific 16S rRNA microbial markers. The second objective was to employ droplet digital PCR (ddPCR) as a novel approach to quantify bacterial species alone and in a mixture of body fluids. Lactobacillus crispatus and Streptococcus salivarius were selected because of high abundance within vaginal fluid and saliva respectively. While Fusobacterium nucleatum and Gardnerella vaginalis, though present in healthy humans, are also frequently found in oral and vaginal infections, respectively. The multiplex PCR assay detected L. crispatus and G. vaginalis in vaginal fluid while F. nucleatum and S. salivarius was detected in saliva. Multiplex PCR detected F. nucleatum, S. salivarius and L. crispatus in mixed body fluid samples while, G. vaginalis was undetected in mixtures containing vaginal fluid. For samples exposed at room temperature for 65 days, L. crispatus and G. vaginalis were detected in vaginal swabs while only S. salivarius was detected in saliva swabs. The limit of detection was 0.06 copies/µl for F. nucleatum (2.5 ×10-9 ng/µl) and S. salivarius (2.5 ×10-6 ng/µl). L. crispatus and G. vaginalis had detection limits of 0.16 copies/µl (2.5 ×10-4 ng/µl) and 0.48 copies/µl (2.5 ×10-7 ng/µl). All 4 bacterial species were detected in mixtures and aged samples by ddPCR. No significant differences were observed in quantity of bacterial markers in saliva and vaginal fluid. The present research reports for the first time the combination of the above four bacterial markers for the detection of saliva and vaginal fluid and highlights the sensitivity of ddPCR for bacterial quantification in pure and mixed body fluids.
Collapse
Affiliation(s)
- Mishka Dass
- Department of Genetics, School of Life Sciences, University of KwaZulu Natal - Westville Campus, Private Bag X 54001, Durban, KwaZulu Natal, South Africa.
| | - Meenu Ghai
- Department of Genetics, School of Life Sciences, University of KwaZulu Natal - Westville Campus, Private Bag X 54001, Durban, KwaZulu Natal, South Africa.
| |
Collapse
|
|
11
|
Chandel DS, Tom WA, Jiang C, Krzyzanowski G, Fernando N, Olou A, Fernando MR. Preanalytical considerations for clinical assays of circulating human miRNA-451a, miRNA-423-5p and miRNA-199a-3p for diagnostic purposes. PLoS One 2024; 19:e0303598. [PMID: 38768135 PMCID: PMC11104614 DOI: 10.1371/journal.pone.0303598] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2024] [Accepted: 04/26/2024] [Indexed: 05/22/2024] Open
Abstract
Circulating miRNA has recently emerged as important biomolecules with potential clinical values as diagnostic markers for several diseases. However, to be used as such, it is critical to accurately quantify miRNAs in the clinic. Yet, preanalytical factors that can affect an error-free quantification of these miRNAs have not been explored. This study aimed at investigating several of these preanalytical factors that may affect the accurate quantification of miRNA-451a, miRNA-423-5p and miRNA-199a-3p in human blood samples. We initially evaluated levels of these three miRNAs in red blood cells (RBCs), white blood cells (WBCs), platelets, and plasma by droplet digital PCR (ddPCR). Next, we monitored miRNA levels in whole blood or platelet rich plasma (PRP) stored at different temperatures for different time periods by ddPCR. We also investigated the effects of hemolysis on miRNA concentrations in platelet-free plasma (PFP). Our results demonstrate that more than 97% of miRNA-451a and miRNA-423-5p in the blood are localized in RBCs, with only trace amounts present in WBCs, platelets, and plasma. Highest amount of the miRNA-199a-3p is present in platelets. Hemolysis had a significant impact on both miRNA-451a and miRNA-423-5p concentrations in plasma, however miRNA-199a levels remain unaffected. Importantly, PRP stored at room temperature (RT) or 4°C showed a statistically significant decrease in miRNA-451a levels, while the other two miRNAs were increased, at days 1, 2, 3 and 7. PFP at RT caused statistically significant steady decline in miRNA-451a and miRNA-423-5p, observed at 12, 24, 36, 48 and 72 hours. Levels of the miRNA-199a-3p in PFP was stable during first 72 hours at RT. PFP stored at -20°C for 7 days showed declining stability of miRNA-451a over time. However, at -80°C miRNA-451a levels were stable up to 7 days. Together, our data indicate that hemolysis and blood storage at RT, 4°C and -20°C may have significant negative effects on the accuracy of circulating miRNA-451a and miRNA-423-5p quantification.
Collapse
Affiliation(s)
- Dinesh S. Chandel
- Molecular Diagnostic Research Laboratory, Center for Sensory Neuroscience, Boys Town National Research Hospital, Omaha, NE, United States of America
| | - Wesley A. Tom
- Molecular Diagnostic Research Laboratory, Center for Sensory Neuroscience, Boys Town National Research Hospital, Omaha, NE, United States of America
| | - Chao Jiang
- Molecular Diagnostic Research Laboratory, Center for Sensory Neuroscience, Boys Town National Research Hospital, Omaha, NE, United States of America
| | - Gary Krzyzanowski
- Molecular Diagnostic Research Laboratory, Center for Sensory Neuroscience, Boys Town National Research Hospital, Omaha, NE, United States of America
| | - Nirmalee Fernando
- Molecular Diagnostic Research Laboratory, Center for Sensory Neuroscience, Boys Town National Research Hospital, Omaha, NE, United States of America
| | - Appolinaire Olou
- Molecular Diagnostic Research Laboratory, Center for Sensory Neuroscience, Boys Town National Research Hospital, Omaha, NE, United States of America
| | - M. Rohan Fernando
- Molecular Diagnostic Research Laboratory, Center for Sensory Neuroscience, Boys Town National Research Hospital, Omaha, NE, United States of America
| |
Collapse
|
|
12
|
Coleman D, Kuwada S. miRNA as a Biomarker for the Early Detection of Colorectal Cancer. Genes (Basel) 2024; 15:338. [PMID: 38540397 PMCID: PMC10969835 DOI: 10.3390/genes15030338] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2024] [Revised: 02/26/2024] [Accepted: 03/03/2024] [Indexed: 06/14/2024] Open
Abstract
MicroRNAs (miRNAs) are short, non-coding RNA segments that can be detected in a variety of clinical samples, including serum, stool, and urine. While miRNAs were initially known for their effect on post-translational gene expression, the last decade of research has shown them to be promising biomarkers for the detection of many types of cancer. This paper explores the use of miRNA detection as a tool for colorectal cancer (CRC) screening. We discuss the current state of miRNA detection, compare it to the existing CRC screening tools, and highlight the advantages and drawbacks of this approach from a clinical and logistical perspective. Our research finds that miRNA-based tests for CRC show great potential, but that widespread clinical adoption will be conditional on future research overcoming key hurdles.
Collapse
Affiliation(s)
- David Coleman
- John A. Burns School of Medicine, University of Hawaii, 651 Ilalo Street, Honolulu, HI 96813, USA
| | - Scott Kuwada
- John A. Burns School of Medicine, University of Hawaii, 651 Ilalo Street, Honolulu, HI 96813, USA
- University of Hawaii Cancer Center, 01 Ilalo Street, Honolulu, HI 96813, USA
| |
Collapse
|
|
13
|
Igder S, Zamani M, Fakher S, Siri M, Ashktorab H, Azarpira N, Mokarram P. Circulating Nucleic Acids in Colorectal Cancer: Diagnostic and Prognostic Value. DISEASE MARKERS 2024; 2024:9943412. [PMID: 38380073 PMCID: PMC10878755 DOI: 10.1155/2024/9943412] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 04/26/2023] [Revised: 01/07/2024] [Accepted: 01/25/2024] [Indexed: 02/22/2024]
Abstract
Colorectal cancer (CRC) is the third most prevalent cancer in the world and the fourth leading cause of cancer-related mortality. DNA (cfDNA/ctDNA) and RNA (cfRNA/ctRNA) in the blood are promising noninvasive biomarkers for molecular profiling, screening, diagnosis, treatment management, and prognosis of CRC. Technological advancements that enable precise detection of both genetic and epigenetic abnormalities, even in minute quantities in circulation, can overcome some of these challenges. This review focuses on testing for circulating nucleic acids in the circulation as a noninvasive method for CRC detection, monitoring, detection of minimal residual disease, and patient management. In addition, the benefits and drawbacks of various diagnostic techniques and associated bioinformatics tools have been detailed.
Collapse
Affiliation(s)
- Somayeh Igder
- Department of Clinical Biochemistry, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Mozhdeh Zamani
- Autophagy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
- Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Shima Fakher
- Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Morvarid Siri
- Autophagy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Hassan Ashktorab
- Department of Medicine, Gastroenterology Division and Cancer Center, Howard University College of Medicine, Washington, DC, USA
| | - Negar Azarpira
- Autophagy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Pooneh Mokarram
- Autophagy Research Center, Department of Biochemistry, Shiraz University of Medical Sciences, Shiraz, Iran
| |
Collapse
|
|
14
|
Isham IM, Najimudeen SM, Cork SC, Gupta A, Abdul-Careem MF. Comparison of quantitative PCR and digital PCR assays for quantitative detection of infectious bronchitis virus (IBV) genome. J Virol Methods 2024; 324:114859. [PMID: 38061673 DOI: 10.1016/j.jviromet.2023.114859] [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: 09/04/2023] [Revised: 11/14/2023] [Accepted: 11/30/2023] [Indexed: 12/22/2023]
Abstract
The quantitative polymerase chain reaction (qPCR) technique is an extensively used molecular tool for the detection and quantification of viral genome load. However, since the qPCR assay is a relative quantification method that relies on an external calibration curve it has a lower assay precision and sensitivity. The digital PCR (dPCR) technique is a good alternative to the qPCR assay as it offers highly precise and direct quantification of viral genome load in samples. In this study, performance characteristics such as the quantification range, sensitivity, precision, and specificity of the dPCR technique was compared to qPCR technique for the detection and quantification of IBV genome loads in serial dilutions of IBV positive plasmid DNA, and IBV infected chicken tissue and swab samples. The quantification range of the qPCR assay was wider than that of the dPCR assay, however dPCR had a higher sensitivity compared to qPCR. The precision of quantification of DNA in plasmid samples in terms of repeatability and reproducibility of results was higher when using the dPCR assay compared to qPCR assay. The quantification results of IBV genome load in infected samples by the qPCR and dPCR assays displayed a high correlation. Hence, our findings suggest that dPCR could be used in avian virology research for improved precision and sensitivity in detection and quantification of viral genome loads.
Collapse
Affiliation(s)
- Ishara M Isham
- Faculty of Veterinary Medicine, University of Calgary, Health Research Innovation Center 2C53, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada
| | - Shahnas M Najimudeen
- Faculty of Veterinary Medicine, University of Calgary, Health Research Innovation Center 2C53, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada
| | - Susan C Cork
- Faculty of Veterinary Medicine, University of Calgary, Health Research Innovation Center 2C53, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada
| | - Ashish Gupta
- Faculty of Veterinary Medicine, University of Calgary, Health Research Innovation Center 2C53, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada
| | - Mohamed Faizal Abdul-Careem
- Faculty of Veterinary Medicine, University of Calgary, Health Research Innovation Center 2C53, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada.
| |
Collapse
|
|
15
|
Wainman LM, Sathyanarayana SH, Lefferts JA. Applications of Digital Polymerase Chain Reaction (dPCR) in Molecular and Clinical Testing. J Appl Lab Med 2024; 9:124-137. [PMID: 38167753 DOI: 10.1093/jalm/jfad103] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2023] [Accepted: 08/15/2023] [Indexed: 01/05/2024]
Abstract
BACKGROUND Digital polymerase chain reaction (dPCR) is an accurate and sensitive molecular method that can be used in clinical diagnostic, prognostic, and predictive tests. The key component of the dPCR method is the partitioning of a single reaction into many thousands of droplets, nanochannels or other nano- or picoliter-sized reactions. This results in high enough sensitivity to detect rare nucleic acid targets and provides an absolute quantification of target sequences or alleles compared to other PCR-based methods. CONTENT An increasing number of dPCR platforms have been introduced commercially in recent years and more are being developed. These platforms differ in the method of partitioning, degree of automation, and multiplexing capabilities but all can be used in similar ways for sensitive and highly accurate quantification of a variety of nucleic acid targets. Currently, clinical applications of dPCR include oncology, microbiology and infectious disease, genetics, and prenatal/newborn screening. Commercially available tests for clinical applications are being developed for variants with diagnostic, prognostic, and therapeutic significance in specific disease types. SUMMARY The power of dPCR technology relies on the partitioning of the reactions and results in increased sensitivity and accuracy compared to qPCR. More recently, the sensitivity of dPCR has been applied to the detection of known variants in cell-free DNA and circulating tumor DNA. Future clinical applications of dPCR include liquid biopsy, treatment resistance detection, screening for minimal residual disease, and monitoring allograft engraftment in transplanted patients.
Collapse
Affiliation(s)
- Lauren M Wainman
- Laboratory for Clinical Genomics and Advanced Technology (CGAT), Department of Pathology and Laboratory Medicine, Dartmouth-Hitchcock Medical Center, Geisel School of Medicine at Dartmouth, Lebanon, NH, United States
| | - Shivaprasad H Sathyanarayana
- Laboratory for Clinical Genomics and Advanced Technology (CGAT), Department of Pathology and Laboratory Medicine, Dartmouth-Hitchcock Medical Center, Geisel School of Medicine at Dartmouth, Lebanon, NH, United States
| | - Joel A Lefferts
- Laboratory for Clinical Genomics and Advanced Technology (CGAT), Department of Pathology and Laboratory Medicine, Dartmouth-Hitchcock Medical Center, Geisel School of Medicine at Dartmouth, Lebanon, NH, United States
| |
Collapse
|
|
16
|
Tavares NT, Lobo J, Bagrodia A. MicroRNAs for detecting occult genitourinary cancer. Curr Opin Urol 2024; 34:20-26. [PMID: 37916954 DOI: 10.1097/mou.0000000000001137] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2023]
Abstract
PURPOSE OF REVIEW Genitourinary (GU) malignancies are a real burden in global health worldwide. Each model has its own clinical challenges, and the early screening and/or detection of occult cancer in follow-up is transversal to all of them. MicroRNAs (miRNAs) have been proposed as minimally invasive liquid biopsy cancer biomarkers, due to their stability and low degradation. RECENT FINDINGS The different GU tumor models are in different stages concerning miRNAs as biomarkers for cancer detection. Testicular germ cell tumors (TGCTs) already have a specific defined target, miR-371a-3p, that has shown high sensitivity and specificity in different clinical settings, and is now in final stages of preanalytical testing before entering the clinic. The other GU malignancies are in a different stage, with many liquid biopsy studies (both in urine and plasma/serum) being currently performed, but there is not an agreeable miRNA or set of miRNAs that is ready to follow the footsteps of miR-371a-3p in TGCTs. SUMMARY Further studies with proper molecular characterization of miRNA profiles of GU malignancies and standardization of sampling, biobanking and formal analysis may aid in the advance and choosing of specific target sets to be used for occult cancer detection.
Collapse
Affiliation(s)
- Nuno Tiago Tavares
- Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Centre Raquel Seruca (Porto.CCC)
- Doctoral Programme in Biomedical Sciences, School of Medicine and Biomedical Sciences, University of Porto (ICBAS-UP)
| | - João Lobo
- Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Centre Raquel Seruca (Porto.CCC)
- Department of Pathology, Portuguese Oncology Institute of Porto/Porto Comprehensive Cancer Centre Raquel Seruca (Porto.CCC)
- Department of Pathology and Molecular Immunology, School of Medicine and Biomedical Sciences, University of Porto (ICBAS-UP), Porto, Portugal
| | - Aditya Bagrodia
- Department of Urology, University of California - San Diego Health, San Diego, California
- Department of Urology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| |
Collapse
|
|
17
|
Maiocchi S, Collins EN, Peterson AR, Alexander KC, McGlamery DJ, Cassidy NA, Ikonomidis JS, Akerman AW. Plasma microrna quantification protocol. VESSEL PLUS 2023; 7:27. [PMID: 38445249 PMCID: PMC10914336 DOI: 10.20517/2574-1209.2023.69] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Indexed: 03/07/2024]
Abstract
MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate translation and are involved in many pathological processes. They have emerged as promising biomarkers for diagnosis of conditions such as aortic aneurysm disease. Quantifying miRNAs in plasma is uniquely challenging because of the lack of standardized reproducible protocols. To facilitate the independent verification of conclusions, it is necessary to provide a thorough disclosure of all pertinent experimental details. In this technical note, we present a comprehensive protocol for quantifying plasma miRNAs using droplet digital PCR. We detail the entire workflow, including blood collection, plasma processing, cryo-storage, miRNA isolation, reverse transcription, droplet generation, PCR amplification, fluorescence reading, and data analysis. We offer comprehensive guidance regarding optimization, assay conditions, expected results, and insight into the troubleshooting of common issues. The stepwise normalization and detailed methodological guide enhance reproducibility. Moreover, multiple portions of this protocol may be automated. The data provided in this technical note is demonstrative of the values typically obtained when following its steps. To facilitate standardization in data reporting, we include a table of expected aortic aneurysm-related miRNA levels in healthy human plasma. This versatile protocol can be easily adapted to quantify most circulating miRNAs in plasma, making it a valuable resource for diagnostic development.
Collapse
Affiliation(s)
- Sophie Maiocchi
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545, USA
| | - Elizabeth N. Collins
- Department of Surgery, Division of Cardiothoracic Surgery, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7065, USA
| | - Andrew R. Peterson
- Department of Surgery, Division of Cardiothoracic Surgery, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7065, USA
| | - Kyle C. Alexander
- Department of Surgery, Division of Cardiothoracic Surgery, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7065, USA
| | - Dalton J. McGlamery
- Department of Surgery, Division of Cardiothoracic Surgery, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7065, USA
| | - Noah A. Cassidy
- University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA
| | - John S. Ikonomidis
- Department of Surgery, Division of Cardiothoracic Surgery, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7065, USA
| | - Adam W. Akerman
- Department of Surgery, Division of Cardiothoracic Surgery, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7065, USA
| |
Collapse
|
|
18
|
Häuser F, Mittler J, Hantal MS, Greulich L, Hermanns M, Shrestha A, Kriege O, Falter T, Immel UD, Herold S, Schuch B, Lackner KJ, Rossmann H, Radsak M. One fits all: a highly sensitive combined ddPCR/pyrosequencing system for the quantification of microchimerism after hematopoietic and solid organ transplantation. Clin Chem Lab Med 2023; 61:1994-2001. [PMID: 37167203 DOI: 10.1515/cclm-2023-0198] [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: 10/11/2022] [Accepted: 04/27/2023] [Indexed: 05/13/2023]
Abstract
OBJECTIVES A combined digital droplet PCR (ddPCR)/pyrosequencing assay system was developed that demonstrated advantages applicable to multiple qualitative and quantitative molecular genetic diagnostic applications. Data for characterizing this combined approach for hematologic stem cell transplantation (HSCT) and allele quantification from graft-derived cell-free (cf) DNA in solid organ transplantation (SOT) is presented. METHODS ddPCR and pyrosequencing assays targeting 32 SNPs/markers were established. ddPCR results from 72 gDNAs of 55 patients after allogeneic HSCT and 107 plasma-cfDNAs of 25 liver transplant recipients were compared with established methods/markers, i.e. short-tandem-repeat PCR and ALT, respectively. RESULTS The ddPCR results were in good agreement with the established marker. The limit of detection was 0.02 % minor allele fraction. The relationship between ddPCR and STR-PCR was linear with R2=0.98 allowing to transfer previously established clinical STR-PCR cut-offs to ddPCR; 50-fold higher sensitivity and a variation coefficient of <2 % enable the use of low DNA concentrations (e.g. pre-sorted cells). ddPCR detected liver allograft injury at least as sensitive as ALT suggesting that ddPCR is a reliable method to monitor the transplant integrity, especially when other biomarkers are lacking (e.g. kidney). CONCLUSIONS Combining pyrosequencing for genotyping and ddPCR for minor allele quantification enhances sensitivity and precision for the patient after HSCT and SOT. The assay is designed for maximum flexibility. It is expected to be suitable for other applications (sample tracking, prenatal diagnostics, etc.).
Collapse
Affiliation(s)
- Friederike Häuser
- Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University Medical Center, Mainz, Germany
| | - Jens Mittler
- Department of General, Visceral, and Transplant Surgery, Johannes Gutenberg University Medical Center, Mainz, Germany
| | - Misra Simge Hantal
- Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University Medical Center, Mainz, Germany
| | - Lilli Greulich
- Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University Medical Center, Mainz, Germany
| | - Martina Hermanns
- Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University Medical Center, Mainz, Germany
| | - Annette Shrestha
- Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University Medical Center, Mainz, Germany
| | - Oliver Kriege
- Department of Medicine III, Johannes Gutenberg University Medical Center, Mainz, Germany
| | - Tanja Falter
- Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University Medical Center, Mainz, Germany
| | - Uta D Immel
- Institute of Legal Medicine, Johannes Gutenberg University Medical Center, Mainz, Germany
| | - Stephanie Herold
- Department of Medicine III, Johannes Gutenberg University Medical Center, Mainz, Germany
| | - Brigitte Schuch
- Department of Medicine III, Johannes Gutenberg University Medical Center, Mainz, Germany
| | - Karl J Lackner
- Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University Medical Center, Mainz, Germany
| | - Heidi Rossmann
- Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University Medical Center, Mainz, Germany
| | - Markus Radsak
- Department of Medicine III, Johannes Gutenberg University Medical Center, Mainz, Germany
| |
Collapse
|
|
19
|
Sequeira JP, Barros-Silva D, Ferreira-Torre P, Salta S, Braga I, Carvalho J, Freitas R, Henrique R, Jerónimo C. OncoUroMiR: Circulating miRNAs for Detection and Discrimination of the Main Urological Cancers Using a ddPCR-Based Approach. Int J Mol Sci 2023; 24:13890. [PMID: 37762193 PMCID: PMC10531069 DOI: 10.3390/ijms241813890] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2023] [Revised: 09/06/2023] [Accepted: 09/07/2023] [Indexed: 09/29/2023] Open
Abstract
The three most common genitourinary malignancies (prostate/kidney/bladder cancers) constitute a substantial proportion of all cancer cases, mainly in the elderly population. Early detection is key to maximizing the patients' survival, but the lack of highly accurate biomarkers that might be used through non-/minimally invasive methods has impaired progress in this domain. Herein, we sought to develop a minimally invasive test to detect and discriminate among those urological cancers based on miRNAs assessment through ddPCR. Plasma samples from 268 patients with renal cell (RCC; n = 119), bladder (BlCa; n = 73), and prostate (PCa; n = 76) carcinomas (UroCancer group), and 74 healthy donors were selected. Hsa-miR-126-3p, hsa-miR-141-3p, hsa-miR-153-5p, hsa-miR-155-5p, hsa-miR-182-5p, hsa-miR-205-5p, and hsa-miR-375-3p levels were assessed. UroCancer cases displayed significantly different circulating hsa-miR-182-5p/hsa-miR-375-3p levels compared to healthy donors. Importantly, the hsa-miR-155-5p/hsa-miR-375-3p panel detected RCC with a high specificity (80.54%) and accuracy (66.04%). Furthermore, the hsa-miR-126-3p/hsa-miR-375-3p panel identified BlCa with a 94.87% specificity and 76.45% NPV whereas higher hsa-miR-126-3p levels were found in PCa patients. We concluded that plasma-derived miRNAs can identify and discriminate among the main genitourinary cancers, with high analytical performance. Although validation in a larger cohort is mandatory, these findings demonstrate that circulating miRNA assessment by ddPCR might provide a new approach for early detection and risk stratification of the most common urological cancers.
Collapse
Affiliation(s)
- José Pedro Sequeira
- Cancer Biology & Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Center Raquel Seruca (Porto.CCC), R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal; (J.P.S.); (D.B.-S.); (P.F.-T.); (S.S.); (I.B.); (J.C.); (R.F.); (R.H.)
- Doctoral Programme in Biomedical Sciences, ICBAS-School of Medicine & Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-513 Porto, Portugal
| | - Daniela Barros-Silva
- Cancer Biology & Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Center Raquel Seruca (Porto.CCC), R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal; (J.P.S.); (D.B.-S.); (P.F.-T.); (S.S.); (I.B.); (J.C.); (R.F.); (R.H.)
| | - Patrícia Ferreira-Torre
- Cancer Biology & Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Center Raquel Seruca (Porto.CCC), R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal; (J.P.S.); (D.B.-S.); (P.F.-T.); (S.S.); (I.B.); (J.C.); (R.F.); (R.H.)
| | - Sofia Salta
- Cancer Biology & Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Center Raquel Seruca (Porto.CCC), R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal; (J.P.S.); (D.B.-S.); (P.F.-T.); (S.S.); (I.B.); (J.C.); (R.F.); (R.H.)
- Doctoral Programme in Molecular Pathology and Genetics, ICBAS-School of Medicine & Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-513 Porto, Portugal
| | - Isaac Braga
- Cancer Biology & Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Center Raquel Seruca (Porto.CCC), R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal; (J.P.S.); (D.B.-S.); (P.F.-T.); (S.S.); (I.B.); (J.C.); (R.F.); (R.H.)
- Department of Urology & Urology Clinics, Portuguese Oncology Institute of Porto (IPOP), R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal
- Doctoral Programme in Medical Sciences, ICBAS-School of Medicine & Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-513 Porto, Portugal
| | - João Carvalho
- Cancer Biology & Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Center Raquel Seruca (Porto.CCC), R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal; (J.P.S.); (D.B.-S.); (P.F.-T.); (S.S.); (I.B.); (J.C.); (R.F.); (R.H.)
- Department of Urology & Urology Clinics, Portuguese Oncology Institute of Porto (IPOP), R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal
- Doctoral Programme in Medical Sciences, ICBAS-School of Medicine & Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-513 Porto, Portugal
| | - Rui Freitas
- Cancer Biology & Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Center Raquel Seruca (Porto.CCC), R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal; (J.P.S.); (D.B.-S.); (P.F.-T.); (S.S.); (I.B.); (J.C.); (R.F.); (R.H.)
- Department of Urology & Urology Clinics, Portuguese Oncology Institute of Porto (IPOP), R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal
- Doctoral Programme in Medical Sciences, ICBAS-School of Medicine & Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-513 Porto, Portugal
| | - Rui Henrique
- Cancer Biology & Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Center Raquel Seruca (Porto.CCC), R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal; (J.P.S.); (D.B.-S.); (P.F.-T.); (S.S.); (I.B.); (J.C.); (R.F.); (R.H.)
- Department of Pathology, Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Center Raquel Seruca (Porto.CCC), R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal
- Department of Pathology and Molecular Immunology, ICBAS–School of Medicine and Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-513 Porto, Portugal
| | - Carmen Jerónimo
- Cancer Biology & Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Center Raquel Seruca (Porto.CCC), R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal; (J.P.S.); (D.B.-S.); (P.F.-T.); (S.S.); (I.B.); (J.C.); (R.F.); (R.H.)
- Department of Pathology, Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Center Raquel Seruca (Porto.CCC), R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal
- Department of Pathology and Molecular Immunology, ICBAS–School of Medicine and Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-513 Porto, Portugal
| |
Collapse
|
|
20
|
Masilamani M, Jawa V, Dai Y, Das R, Park A, Lamba M, Wu F, Zheng X, Lu E, Gleason C, Mack T, Mora J, Surapaneni S. Bioanalytical Methods for Characterization of CAR-T Cellular Kinetics: Comparison of PCR Assays and Matrices. Clin Pharmacol Ther 2023; 114:664-672. [PMID: 37422675 DOI: 10.1002/cpt.2991] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2023] [Accepted: 06/27/2023] [Indexed: 07/10/2023]
Abstract
Recently, multiple chimeric antigen receptor T-cell (CAR-T)-based therapies have been approved for treating hematological malignancies, targeting CD19 and B-cell maturation antigen. Unlike protein or antibody therapies, CAR-T therapies are "living cell" therapies whose pharmacokinetics are characterized by expansion, distribution, contraction, and persistence. Therefore, this unique modality requires a different approach for quantitation compared with conventional ligand binding assays implemented for most biologics. Cellular (flow cytometry) or molecular assays (polymerase chain reaction (PCR)) can be deployed with each having unique advantages and disadvantages. In this article, we describe the molecular assays utilized: quantitative PCR (qPCR), which was the initial platform used to estimate transgene copy numbers and more recently droplet digital PCR (ddPCR) which quantitates the absolute copy numbers of CAR transgene. The comparability of the two methods in patient samples and of each method across different matrices (isolated CD3+ T-cells or whole blood) was also performed. The results show a good correlation between qPCR and ddPCR for the amplification of same gene in clinical samples from a CAR-T therapy trial. In addition, our studies show that the qPCR-based amplification of transgene levels was well-correlated, independent of DNA sources (either CD3+ T-cells or whole blood). Our results also highlight that ddPCR can be a better platform for monitoring samples at the early phase of CAR-T dosing prior to expansion and during long-term monitoring as they can detect samples with very low copy numbers with high sensitivity, in addition to easier implementation and sample logistics.
Collapse
Affiliation(s)
- Madhan Masilamani
- Department of Clinical Pharmacology, Pharmacometrics, Disposition, and Bioanalysis, Bristol Myers Squibb, Lawrenceville, New Jersey, USA
| | - Vibha Jawa
- Department of Clinical Pharmacology, Pharmacometrics, Disposition, and Bioanalysis, Bristol Myers Squibb, Lawrenceville, New Jersey, USA
| | - Yanshan Dai
- Department of Clinical Pharmacology, Pharmacometrics, Disposition, and Bioanalysis, Bristol Myers Squibb, Lawrenceville, New Jersey, USA
| | - Romita Das
- Department of Clinical Pharmacology, Pharmacometrics, Disposition, and Bioanalysis, Bristol Myers Squibb, Lawrenceville, New Jersey, USA
| | - Alice Park
- Department of Clinical Pharmacology, Pharmacometrics, Disposition, and Bioanalysis, Bristol Myers Squibb, Lawrenceville, New Jersey, USA
| | - Manisha Lamba
- Department of Clinical Pharmacology, Pharmacometrics, Disposition, and Bioanalysis, Bristol Myers Squibb, Lawrenceville, New Jersey, USA
| | - Fan Wu
- Department of Clinical Pharmacology, Pharmacometrics, Disposition, and Bioanalysis, Bristol Myers Squibb, Lawrenceville, New Jersey, USA
| | - Xirong Zheng
- Department of Clinical Pharmacology, Pharmacometrics, Disposition, and Bioanalysis, Bristol Myers Squibb, Lawrenceville, New Jersey, USA
| | - Edwin Lu
- Global Biometrics and Data Sciences, Bristol Myers Squibb, Lawrenceville, New Jersey, USA
| | - Carol Gleason
- Global Biometrics and Data Sciences, Bristol Myers Squibb, Lawrenceville, New Jersey, USA
| | - Tim Mack
- Department of Clinical Pharmacology, Pharmacometrics, Disposition, and Bioanalysis, Bristol Myers Squibb, Lawrenceville, New Jersey, USA
| | - Johanna Mora
- Department of Clinical Pharmacology, Pharmacometrics, Disposition, and Bioanalysis, Bristol Myers Squibb, Lawrenceville, New Jersey, USA
| | - Sekhar Surapaneni
- Department of Clinical Pharmacology, Pharmacometrics, Disposition, and Bioanalysis, Bristol Myers Squibb, Lawrenceville, New Jersey, USA
| |
Collapse
|
|
21
|
Tariq L, Arafah A, Sehar N, Ali A, Khan A, Rasool I, Rashid SM, Ahmad SB, Beigh S, Dar TUH, Rehman MU. Novel insights on perils and promises of miRNA in understanding colon cancer metastasis and progression. Med Oncol 2023; 40:282. [PMID: 37639075 DOI: 10.1007/s12032-023-02099-2] [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: 05/23/2023] [Accepted: 06/19/2023] [Indexed: 08/29/2023]
Abstract
Colorectal cancer (CRC) is the third highest frequent malignancy and ultimate critical source of cancer-associated mortality around the world. Regardless of latest advances in molecular and surgical targeted medicines that have increased remedial effects in CRC patients, the 5-year mortality rate for CRC patients remains dismally low. Evidence suggests that microRNAs (miRNAs) execute an essential part in the development and spread of CRC. The miRNAs are a type of short non-coding RNA that exhibited to control the appearance of tumor suppressor genes and oncogenes. miRNA expression profiling is already being utilized in clinical practice as analytical and prognostic biomarkers to evaluate cancer patients' tumor genesis, advancement, and counteraction to drugs. By modulating their target genes, dysregulated miRNAs are linked to malignant characteristics (e.g., improved proliferative and invasive capabilities, cell cycle aberration, evasion of apoptosis, and promotion of angiogenesis). This review presents an updated summary of circulatory miRNAs, tumor-suppressive and oncogenic miRNAs, and the potential reasons for dysregulated miRNAs in CRC. Further we will explore the critical role of miRNAs in CRC drug resistance.
Collapse
Affiliation(s)
- Lubna Tariq
- Department of Biotechnology, Baba Ghulam Shah Badshah University, Rajouri, Jammu and Kashmir, 183254, India
| | - Azher Arafah
- Department of Clinical Pharmacy, College of Pharmacy, King Saud University, 11451, Riyadh, Saudi Arabia
| | - Nouroz Sehar
- Centre for Translational and Clinical Research, School of Chemical & Life Sciences, Jamia Hamdard, New Delhi, 110062, India
| | - Aarif Ali
- Division of Veterinary Biochemistry, Faculty of Veterinary Science and Animal Husbandry, SKUAST-Kashmir, Alusteng, Shuhama, Srinagar, Jammu and Kashmir, 190006, India
| | - Andleeb Khan
- Department of Pharmacology and Toxicology, College of Pharmacy, Jazan University, 45142, Jazan, Saudi Arabia
| | - Iyman Rasool
- Department of Pathology, Government Medical College (GMC-Srinagar), Karanagar, Srinagar, Jammu and Kashmir, 190006, India
| | - Shahzada Mudasir Rashid
- Division of Veterinary Biochemistry, Faculty of Veterinary Science and Animal Husbandry, SKUAST-Kashmir, Alusteng, Shuhama, Srinagar, Jammu and Kashmir, 190006, India
| | - Sheikh Bilal Ahmad
- Division of Veterinary Biochemistry, Faculty of Veterinary Science and Animal Husbandry, SKUAST-Kashmir, Alusteng, Shuhama, Srinagar, Jammu and Kashmir, 190006, India
| | - Saba Beigh
- Department of Public Health, Faculty of Applied Medical Science, Al Baha University, 65431, Al Baha, Saudi Arabia
| | - Tanveer Ul Hassan Dar
- Department of Biotechnology, Baba Ghulam Shah Badshah University, Rajouri, Jammu and Kashmir, 183254, India
| | - Muneeb U Rehman
- Department of Clinical Pharmacy, College of Pharmacy, King Saud University, 11451, Riyadh, Saudi Arabia.
| |
Collapse
|
|
22
|
Bache M, Kadler F, Struck O, Medenwald D, Ostheimer C, Güttler A, Keßler J, Kappler M, Riemann A, Thews O, Seliger B, Vordermark D. Correlation between Circulating miR-16, miR-29a, miR-144 and miR-150, and the Radiotherapy Response and Survival of Non-Small-Cell Lung Cancer Patients. Int J Mol Sci 2023; 24:12835. [PMID: 37629015 PMCID: PMC10454434 DOI: 10.3390/ijms241612835] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2023] [Revised: 08/10/2023] [Accepted: 08/12/2023] [Indexed: 08/27/2023] Open
Abstract
Despite the success of current therapy concepts, patients with advanced non-small-cell lung cancer (NSCLC) still have a very poor prognosis. Therefore, biological markers are urgently needed, which allow the assessment of prognosis, or prediction of the success of therapy or resistance in this disease. Circulating microRNAs (miRs) have potential as biomarkers for the prognosis and prediction of response to therapy in cancer patients. Based on recent evidence that circulating miR-16, miR-29a, miR-144 and miR-150 can be regulated by ionizing radiation, the concentration of these four miRs was assessed in the plasma of NSCLC patients at different time points of radiotherapy by digital droplet PCR (ddPCR). Furthermore, their impact on patients' prognosis was evaluated. The mean plasma levels of miR-16, miR-29a, miR-144 and miR-150 significantly differed intra- and inter-individually, and during therapy in NSCLC patients, but showed a strong positive correlation. The individual plasma levels of miR-16, miR-29a and miR-144 had prognostic value in NSCLC patients during or at the end of radiotherapy in Cox's regression models. NSCLC patients with low levels of these three miRs at the end of radiotherapy had the worst prognosis. However, miR-150 plasma levels and treatment-dependent changes were not predictive. In conclusion, circulating miR-16, miR-29a and miR-144, but not miR-150, have a prognostic value in NSCLC patients undergoing radiotherapy.
Collapse
Affiliation(s)
- Matthias Bache
- Department of Radiotherapy, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06120 Halle, Germany; (F.K.); (O.S.); (D.M.); (A.G.); (J.K.); (D.V.)
| | - Frauke Kadler
- Department of Radiotherapy, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06120 Halle, Germany; (F.K.); (O.S.); (D.M.); (A.G.); (J.K.); (D.V.)
| | - Olivia Struck
- Department of Radiotherapy, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06120 Halle, Germany; (F.K.); (O.S.); (D.M.); (A.G.); (J.K.); (D.V.)
- Department of Radiology, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06120 Halle, Germany
| | - Daniel Medenwald
- Department of Radiotherapy, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06120 Halle, Germany; (F.K.); (O.S.); (D.M.); (A.G.); (J.K.); (D.V.)
| | - Christian Ostheimer
- Department of Radiotherapy, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06120 Halle, Germany; (F.K.); (O.S.); (D.M.); (A.G.); (J.K.); (D.V.)
| | - Antje Güttler
- Department of Radiotherapy, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06120 Halle, Germany; (F.K.); (O.S.); (D.M.); (A.G.); (J.K.); (D.V.)
| | - Jacqueline Keßler
- Department of Radiotherapy, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06120 Halle, Germany; (F.K.); (O.S.); (D.M.); (A.G.); (J.K.); (D.V.)
| | - Matthias Kappler
- Department of Oral and Maxillofacial Plastic Surgery, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06120 Halle, Germany;
| | - Anne Riemann
- Julius Bernstein Institute of Physiology, Martin Luther University Halle-Wittenberg, Magdeburger Str. 6, 06112 Halle, Germany; (A.R.); (O.T.)
| | - Oliver Thews
- Julius Bernstein Institute of Physiology, Martin Luther University Halle-Wittenberg, Magdeburger Str. 6, 06112 Halle, Germany; (A.R.); (O.T.)
| | - Barbara Seliger
- Medical Faculty, Martin Luther University Halle-Wittenberg, Magdeburger Str. 16, 06112 Halle, Germany;
- Institute for Translational Immunology, Brandenburg Medical School “Theodor Fontane”, 14770 Brandenburg, Germany
- Fraunhofer Institute for Cell Therapy and Immunology, 04103 Leipzig, Germany
| | - Dirk Vordermark
- Department of Radiotherapy, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06120 Halle, Germany; (F.K.); (O.S.); (D.M.); (A.G.); (J.K.); (D.V.)
| |
Collapse
|
|
23
|
Mai HT, Vanness BC, Linz TH. Reverse transcription-free digital-quantitative-PCR for microRNA analysis. Analyst 2023; 148:3019-3027. [PMID: 37264955 PMCID: PMC10318481 DOI: 10.1039/d3an00351e] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/03/2023]
Abstract
MicroRNAs (miRNAs) are non-coding RNA sequences that regulate many biological processes and have become central targets of biomedical research. However, their naturally low abundances in biological samples necessitates the development of sensitive analytical techniques to conduct routine miRNA measurements in research laboratories. Digital PCR has the potential to meet this need because of its single-molecule detection capabilities, but PCR analyses of miRNAs are slowed by the ligation and reverse transcription steps first required to prepare samples. This report describes the development of a method to rapidly quantify miRNA in digital microwell arrays using base-stacking digital-quantitative-PCR (BS-dqPCR). BS-dqPCR expedites miRNA measurements by eliminating the need for ligation and reverse transcription steps, which reduces the time and cost compared to conventional miRNA PCR analyses. Under standard PCR thermocycling conditions, digital signals from miRNA samples were lower than expected, while signals from blanks were high. Therefore, a novel asymmetric thermocycling program was developed that maximized on-target signal from miRNA while minimizing non-specific amplification. The analytical response of BS-dqPCR was then evaluated over a range of miRNA concentrations. The digital PCR dimension increased in signal with increasing miRNA copy numbers. When the digital signal saturated, the quantitative PCR dimension readily discerned miRNA copy number differences. Overall, BS-dqPCR provides rapid, high-sensitivity measurements of miRNA over a wide dynamic range, which demonstrates its utility for routine miRNA analyses.
Collapse
Affiliation(s)
- Hao T Mai
- Department of Chemistry, Wayne State University, 5101 Cass Ave, Detroit, MI 48202, USA.
| | - Brice C Vanness
- Department of Chemistry, Wayne State University, 5101 Cass Ave, Detroit, MI 48202, USA.
| | - Thomas H Linz
- Department of Chemistry, Wayne State University, 5101 Cass Ave, Detroit, MI 48202, USA.
| |
Collapse
|
|
24
|
Roy JW, Wajnberg G, Ouellette A, Boucher JE, Lacroix J, Chacko S, Ghosh A, Ouellette RJ, Lewis SM. Small RNA sequencing analysis of peptide-affinity isolated plasma extracellular vesicles distinguishes pancreatic cancer patients from non-affected individuals. Sci Rep 2023; 13:9251. [PMID: 37286718 DOI: 10.1038/s41598-023-36370-3] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2023] [Accepted: 06/02/2023] [Indexed: 06/09/2023] Open
Abstract
Pancreatic ductal adenocarcinoma (PDAC) has a high fatality rate, mainly due to its asymptomatic nature until late-stage disease and therefore delayed diagnosis that leads to a lack of timely treatment intervention. Consequently, there is a significant need for better methods to screen populations that are at high risk of developing PDAC. Such advances would result in earlier diagnosis, more treatment options, and ultimately better outcomes for patients. Several recent studies have applied the concept of liquid biopsy, which is the sampling of a biofluid (such as blood plasma) for the presence of disease biomarkers, to develop screening approaches for PDAC; several of these studies have focused on analysis of extracellular vesicles (EVs) and their cargoes. While these studies have identified many potential biomarkers for PDAC that are present within EVs, their application to clinical practice is hindered by the lack of a robust, reproducible method for EV isolation and analysis that is amenable to a clinical setting. Our previous research has shown that the Vn96 synthetic peptide is indeed a robust and reproducible method for EV isolation that has the potential to be used in a clinical setting. We have therefore chosen to investigate the utility of the Vn96 synthetic peptide for this isolation of EVs from human plasma and the subsequent detection of small RNA biomarkers of PDAC by Next-generation sequencing (NGS) analysis. We find that analysis of small RNA from Vn96-isolated EVs permits the discrimination of PDAC patients from non-affected individuals. Moreover, analyses of all small RNA species, miRNAs, and lncRNA fragments are most effective at segregating PDAC patients from non-affected individuals. Several of the identified small RNA biomarkers have been previously associated with and/or characterized in PDAC, indicating the validity of our findings, whereas other identified small RNA biomarkers may have novel roles in PDAC or cancer in general. Overall, our results provide a basis for a clinically-amendable detection and/or screening strategy for PDAC using a liquid biopsy approach that relies on Vn96-mediated isolation of EVs from plasma.
Collapse
Affiliation(s)
- Jeremy W Roy
- Atlantic Cancer Research Institute, Moncton, NB, Canada
- Beatrice Hunter Cancer Research Institute, Halifax, NS, Canada
| | | | | | | | | | - Simi Chacko
- Atlantic Cancer Research Institute, Moncton, NB, Canada
| | - Anirban Ghosh
- Atlantic Cancer Research Institute, Moncton, NB, Canada
| | - Rodney J Ouellette
- Atlantic Cancer Research Institute, Moncton, NB, Canada
- Beatrice Hunter Cancer Research Institute, Halifax, NS, Canada
- Department of Chemistry and Biochemistry, Université de Moncton, Moncton, NB, Canada
- Dr. Georges-L.-Dumont University Hospital Centre, Moncton, NB, Canada
| | - Stephen M Lewis
- Atlantic Cancer Research Institute, Moncton, NB, Canada.
- Beatrice Hunter Cancer Research Institute, Halifax, NS, Canada.
- Department of Chemistry and Biochemistry, Université de Moncton, Moncton, NB, Canada.
| |
Collapse
|
|
25
|
Ciobanu CG, Nucă I, Popescu R, Antoci LM, Caba L, Ivanov AV, Cojocaru KA, Rusu C, Mihai CT, Pânzaru MC. Narrative Review: Update on the Molecular Diagnosis of Fragile X Syndrome. Int J Mol Sci 2023; 24:9206. [PMID: 37298158 PMCID: PMC10252420 DOI: 10.3390/ijms24119206] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2023] [Revised: 04/30/2023] [Accepted: 05/20/2023] [Indexed: 06/12/2023] Open
Abstract
The diagnosis and management of fragile X syndrome (FXS) have significantly improved in the last three decades, although the current diagnostic techniques are not yet able to precisely identify the number of repeats, methylation status, level of mosaicism, and/or the presence of AGG interruptions. A high number of repeats (>200) in the fragile X messenger ribonucleoprotein 1 gene (FMR1) results in hypermethylation of promoter and gene silencing. The actual molecular diagnosis is performed using a Southern blot, TP-PCR (Triplet-Repeat PCR), MS-PCR (Methylation-Specific PCR), and MS-MLPA (Methylation-Specific MLPA) with some limitations, with multiple assays being necessary to completely characterise a patient with FXS. The actual gold standard diagnosis uses Southern blot; however, it cannot accurately characterise all cases. Optical genome mapping is a new technology that has also been developed to approach the diagnosis of fragile X syndrome. Long-range sequencing represented by PacBio and Oxford Nanopore has the potential to replace the actual diagnosis and offers a complete characterization of molecular profiles in a single test. The new technologies have improved the diagnosis of fragile X syndrome and revealed unknown aberrations, but they are a long way from being used routinely in clinical practice.
Collapse
Affiliation(s)
- Cristian-Gabriel Ciobanu
- Medical Genetics Department, Faculty of Medicine, “Grigore T. Popa” University of Medicine and Pharmacy, University Street No 16, 700115 Iasi, Romania; (C.-G.C.)
| | - Irina Nucă
- Medical Genetics Department, Faculty of Medicine, “Grigore T. Popa” University of Medicine and Pharmacy, University Street No 16, 700115 Iasi, Romania; (C.-G.C.)
- Investigatii Medicale Praxis, St. Moara de Vant No 35, 700376 Iasi, Romania
| | - Roxana Popescu
- Medical Genetics Department, Faculty of Medicine, “Grigore T. Popa” University of Medicine and Pharmacy, University Street No 16, 700115 Iasi, Romania; (C.-G.C.)
- Medical Genetics Department, “Saint Mary” Emergency Children’s Hospital, St. Vasile Lupu No 62, 700309 Iasi, Romania
| | - Lucian-Mihai Antoci
- Medical Genetics Department, Faculty of Medicine, “Grigore T. Popa” University of Medicine and Pharmacy, University Street No 16, 700115 Iasi, Romania; (C.-G.C.)
| | - Lavinia Caba
- Medical Genetics Department, Faculty of Medicine, “Grigore T. Popa” University of Medicine and Pharmacy, University Street No 16, 700115 Iasi, Romania; (C.-G.C.)
| | - Anca Viorica Ivanov
- Pediatrics Department, “Grigore T. Popa” University of Medicine and Pharmacy, University Street No 16, 700115 Iasi, Romania
| | - Karina-Alexandra Cojocaru
- Department of Biochemistry, Faculty of Dental Medicine, “Grigore T. Popa” University of Medicine and Pharmacy, University Street No 16, 700115 Iasi, Romania
| | - Cristina Rusu
- Medical Genetics Department, Faculty of Medicine, “Grigore T. Popa” University of Medicine and Pharmacy, University Street No 16, 700115 Iasi, Romania; (C.-G.C.)
- Medical Genetics Department, “Saint Mary” Emergency Children’s Hospital, St. Vasile Lupu No 62, 700309 Iasi, Romania
| | | | - Monica-Cristina Pânzaru
- Medical Genetics Department, Faculty of Medicine, “Grigore T. Popa” University of Medicine and Pharmacy, University Street No 16, 700115 Iasi, Romania; (C.-G.C.)
- Medical Genetics Department, “Saint Mary” Emergency Children’s Hospital, St. Vasile Lupu No 62, 700309 Iasi, Romania
| |
Collapse
|
|
26
|
Di Mauro G, Frontini F, Torreggiani E, Iaquinta MR, Caselli A, Mazziotta C, Esposito V, Mazzoni E, Libener R, Grosso F, Maconi A, Martini F, Bononi I, Tognon M. Epigenetic investigation into circulating microRNA 197-3p in sera from patients affected by malignant pleural mesothelioma and workers ex-exposed to asbestos. Sci Rep 2023; 13:6501. [PMID: 37081052 PMCID: PMC10119131 DOI: 10.1038/s41598-023-33116-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2023] [Accepted: 04/07/2023] [Indexed: 04/22/2023] Open
Abstract
The epigenetic role of microRNAs is established at both physiological and pathological levels. Dysregulated miRNAs and their targets appear to be a promising approach for innovative anticancer therapies. In our previous study, circulating miR-197-3p tested dysregulated in workers ex-exposed to asbestos (WEA). Herein, an epigenetic investigation on this circulating miRNA was carried out in sera from malignant pleural mesothelioma (MPM) patients. MiR-197-3p was quantified in MPM (n = 75) sera and comparatively analyzed to WEA (n = 75) and healthy subject (n = 75) sera, using ddPCR and RT-qPCR techniques. Clinicopathological characteristics, occupational, non-occupational information and overall survival (OS) were evaluated in correlation studies. MiR-197-3p levels, analyzed by ddPCR, were significantly higher in MPM than in WEA cohort, with a mean copies/µl of 981.7 and 525.01, respectively. Consistently, RT-qPCR showed higher miR-197-3p levels in sera from MPM with a mean copies/µl of 603.7, compared to WEA with 336.1 copies/µl. OS data were significantly associated with histologic subtype and pleurectomy. Circulating miR-197-3p is proposed as a new potential biomarker for an early diagnosis of the MPM onset. Indeed, miR-197-3p epigenetic investigations along with chest X-ray, computed tomography scan and spirometry could provide relevant information useful to reach an early and effective diagnosis for MPM.
Collapse
Affiliation(s)
- Giulia Di Mauro
- Department of Medical Sciences, Laboratories of Cell Biology and Molecular Genetics, School of Medicine, University of Ferrara, 64B, Fossato di Mortara Street, 44121, Ferrara, Italy
| | - Francesca Frontini
- Department of Medical Sciences, Laboratories of Cell Biology and Molecular Genetics, School of Medicine, University of Ferrara, 64B, Fossato di Mortara Street, 44121, Ferrara, Italy
| | - Elena Torreggiani
- Department of Medical Sciences, Laboratories of Cell Biology and Molecular Genetics, School of Medicine, University of Ferrara, 64B, Fossato di Mortara Street, 44121, Ferrara, Italy
| | - Maria Rosa Iaquinta
- Department of Medical Sciences, Laboratories of Cell Biology and Molecular Genetics, School of Medicine, University of Ferrara, 64B, Fossato di Mortara Street, 44121, Ferrara, Italy
| | - Andrea Caselli
- Department of Medical Sciences, Laboratories of Cell Biology and Molecular Genetics, School of Medicine, University of Ferrara, 64B, Fossato di Mortara Street, 44121, Ferrara, Italy
| | - Chiara Mazziotta
- Department of Medical Sciences, Laboratories of Cell Biology and Molecular Genetics, School of Medicine, University of Ferrara, 64B, Fossato di Mortara Street, 44121, Ferrara, Italy
| | - Valentina Esposito
- Department of Medical Sciences, Laboratories of Cell Biology and Molecular Genetics, School of Medicine, University of Ferrara, 64B, Fossato di Mortara Street, 44121, Ferrara, Italy
| | - Elisa Mazzoni
- Department of Chemical, Pharmaceutical and Agricultural Sciences-DOCPAS, University of Ferrara, 44121, Ferrara, Italy
| | - Roberta Libener
- Research Training and Innovation Infrastructure - Department of Integrated Research and Innovation Activities (DAIRI), AO SS. Antonio e Biagio e Cesare Arrigo, 15121, Alessandria, Italy
| | - Federica Grosso
- Mesothelioma Unit, AO SS. Antonio e Biagio e Cesare Arrigo, 15121, Alessandria, Italy
| | - Antonio Maconi
- Research Training and Innovation Infrastructure - Department of Integrated Research and Innovation Activities (DAIRI), AO SS. Antonio e Biagio e Cesare Arrigo, 15121, Alessandria, Italy
| | - Fernanda Martini
- Department of Medical Sciences, Laboratories of Cell Biology and Molecular Genetics, School of Medicine, University of Ferrara, 64B, Fossato di Mortara Street, 44121, Ferrara, Italy
| | - Ilaria Bononi
- Department of Translational Medicine and for Romagna, University of Ferrara, 70, Fossato di Mortara Street, 44121, Ferrara, Italy.
| | - Mauro Tognon
- Department of Medical Sciences, Laboratories of Cell Biology and Molecular Genetics, School of Medicine, University of Ferrara, 64B, Fossato di Mortara Street, 44121, Ferrara, Italy.
| |
Collapse
|
|
27
|
Bitenc M, Grebstad Tune B, Melheim M, Atneosen-Åsegg M, Lai X, Rajar P, Solberg R, Baumbusch LO. Assessing nuclear versus mitochondrial cell-free DNA (cfDNA) by qRT-PCR and droplet digital PCR using a piglet model of perinatal asphyxia. Mol Biol Rep 2023; 50:1533-1544. [PMID: 36512170 PMCID: PMC9889441 DOI: 10.1007/s11033-022-08135-0] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2022] [Accepted: 11/17/2022] [Indexed: 12/15/2022]
Abstract
BACKGROUND Since the discovery more than half a century ago, cell-free DNA (cfDNA) has become an attractive objective in multiple diagnostic, prognostic, and monitoring settings. However, despite the increasing number of cfDNA applications in liquid biopsies, we still lack a comprehensive understanding of the nature of cfDNA including optimal assessment. In the presented study, we continued testing and validation of common techniques for cfDNA extraction and quantification (qRT-PCR or droplet digital PCR) of nuclear- and mitochondrial cfDNA (ncfDNA and mtcfDNA) in blood, using a piglet model of perinatal asphyxia to determine potential temporal and quantitative changes at the levels of cfDNA. METHODS AND RESULTS Newborn piglets (n = 19) were either exposed to hypoxia (n = 11) or were part of the sham-operated control group (n = 8). Blood samples were collected at baseline (= start) and at the end of hypoxia or at 40-45 min for the sham-operated control group. Applying the qRT-PCR method, ncfDNA concentrations in piglets exposed to hypoxia revealed an increasing trend from 7.1 ng/ml to 9.5 ng/ml for HK2 (hexokinase 2) and from 4.6 ng/ml to 7.9 ng/ml for β-globulin, respectively, whereas the control animals showed a more balanced profile. Furthermore, median levels of mtcfDNA were much higher in comparison to ncfDNA, but without significant differences between intervention versus the control group. CONCLUSIONS Both, qRT-PCR and the droplet digital PCR technique identified overall similar patterns for the concentration changes of cfDNA; but, the more sensitive digital PCR methodology might be required to identify minimal responses.
Collapse
Affiliation(s)
- Marie Bitenc
- Department of Pediatric Research, Division of Paediatric and Adolescent Medicine, Oslo University Hospital Rikshospitalet, Postbox 4950, 0424, Nydalen, Oslo, Norway
| | - Benedicte Grebstad Tune
- Department of Pediatric Research, Division of Paediatric and Adolescent Medicine, Oslo University Hospital Rikshospitalet, Postbox 4950, 0424, Nydalen, Oslo, Norway
- Institute of Clinical Medicine, University of Oslo, Oslo, Norway
| | - Maria Melheim
- Department of Pediatric Research, Division of Paediatric and Adolescent Medicine, Oslo University Hospital Rikshospitalet, Postbox 4950, 0424, Nydalen, Oslo, Norway
| | | | - Xiaoran Lai
- Oslo Centre for Biostatistics and Epidemiology, Faculty of Medicine, University of Oslo, Oslo, Norway
| | - Polona Rajar
- Department of Neonatal Intensive Care, Division of Paediatric and Adolescent Medicine, Oslo University Hospital Ullevål, Oslo, Norway
- Institute of Oral Biology, University of Oslo, Oslo, Norway
| | - Rønnaug Solberg
- Department of Pediatric Research, Division of Paediatric and Adolescent Medicine, Oslo University Hospital Rikshospitalet, Postbox 4950, 0424, Nydalen, Oslo, Norway
- Department of Pediatrics, Vestfold Hospital Trust, Tønsberg, Norway
| | - Lars Oliver Baumbusch
- Department of Pediatric Research, Division of Paediatric and Adolescent Medicine, Oslo University Hospital Rikshospitalet, Postbox 4950, 0424, Nydalen, Oslo, Norway.
| |
Collapse
|
|
28
|
Zamboni C, Zamarian V, Stefanello D, Ferrari R, Auletta L, Milanesi S, Mauri S, Grieco V, Ceciliani F, Lecchi C. Plasma small extracellular vesicles from dogs affected by cutaneous mast cell tumors deliver high levels of miR-21-5p. Front Vet Sci 2023; 9:1083174. [PMID: 36704706 PMCID: PMC9871458 DOI: 10.3389/fvets.2022.1083174] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2022] [Accepted: 12/13/2022] [Indexed: 01/11/2023] Open
Abstract
Small extracellular vesicles (sEV) are a class of extracellular vesicles (30-150 nm), delivering molecules including proteins, metabolites, and microRNAs (miRNAs), involved in physiological intercellular crosstalk and disease pathogenesis. The present pilot study aims are (I) to develop an easy and fast protocol for the isolation of sEV from plasma of mast cell tumor (MCT)-affected dogs; (II) to evaluate if miR-21-5p (sEV-miR-21-5p), a miRNA overexpressed by MCT, is associated with sEV. Seventeen dogs have been enrolled in the study: 4 healthy and 13 (6 with and 7 without nodal metastasis) MCT-affected dogs. sEV were isolated using size exclusion chromatography (SEC) (IZON column 35nm) and were characterized by Western blot, Nanoparticle tracking analysis, and transmission electron microscopy. sEV-miR-21-5p was quantified using digital PCR. sEV expressed the specific markers CD9 and TSG101, and a marker of mast cell tryptase. The sEV mean concentration and size were 2.68E + 10 particles/ml, and 99.6 nm, 2.89E + 10 particles/ml and 101.7 nm, and 3.21E + 10 particles/ml and 124 nm in non-metastatic, nodal metastatic, and healthy samples, respectively. The comparative analysis demonstrated that the level of sEV-miR-21-5p was significantly higher in dogs with nodal metastasis compared to healthy (P = 0.038) and without nodal metastasis samples (P = 0.007). In conclusion, the present work demonstrated that a pure population of sEV can be isolated from the plasma of MCT-affected dogs using the SEC approach and that the level of sEV-miR-21-5p is higher in nodal metastatic MCT-affected dogs compared with healthy and MCT-affected dogs without nodal involvement.
Collapse
Affiliation(s)
- Clarissa Zamboni
- Dipartimento di Medicina Veterinaria e Scienze Animali, Università degli Studi di Milano, Milan, Italy
| | - Valentina Zamarian
- Dipartimento di Medicina Veterinaria e Scienze Animali, Università degli Studi di Milano, Milan, Italy,Diabetes Research Institute, IRCCS San Raffaele Hospital, Milan, Italy
| | - Damiano Stefanello
- Dipartimento di Medicina Veterinaria e Scienze Animali, Università degli Studi di Milano, Milan, Italy
| | - Roberta Ferrari
- Dipartimento di Medicina Veterinaria e Scienze Animali, Università degli Studi di Milano, Milan, Italy
| | - Luigi Auletta
- Dipartimento di Medicina Veterinaria e Scienze Animali, Università degli Studi di Milano, Milan, Italy
| | - Samantha Milanesi
- Leukocytes Biology Group, IRCCS Humanitas Clinical and Research Center, Milan, Italy,Department of Medical Biotechnologies and Translational Medicine, Università degli Studi di Milano, Milan, Italy
| | - Samuele Mauri
- Dipartimento di Medicina Veterinaria e Scienze Animali, Università degli Studi di Milano, Milan, Italy
| | - Valeria Grieco
- Dipartimento di Medicina Veterinaria e Scienze Animali, Università degli Studi di Milano, Milan, Italy
| | - Fabrizio Ceciliani
- Dipartimento di Medicina Veterinaria e Scienze Animali, Università degli Studi di Milano, Milan, Italy
| | - Cristina Lecchi
- Dipartimento di Medicina Veterinaria e Scienze Animali, Università degli Studi di Milano, Milan, Italy,*Correspondence: Cristina Lecchi ✉
| |
Collapse
|
|
29
|
Mestry C, Ashavaid TF, Shah SA. Key methodological challenges in detecting circulating miRNAs in different biofluids. Ann Clin Biochem 2023; 60:14-26. [PMID: 36113172 DOI: 10.1177/00045632221129778] [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: 01/20/2023]
Abstract
The technological advancement in diagnostic techniques has immensely improved the capability of predicting disease progression. Yet, there is a great interest in developing newer biomarkers that can enhance disease risk prediction thereby minimising the associated morbidity and mortality. Circulating miRNAs, a non-coding RNA molecule, are critical regulators in the pathophysiology of various complex multifactorial diseases. In recent years, circulating miRNAs have been enormously studied and are considered as an emerging biomarker due to their easy accessibility, stability, and detection by sequence-specific amplification methods. However, there is a distinct lack of consensus regarding the preanalytical factors such as preferred sample selection, methodological aspects, etc that may independently or together influence the detection of circulating miRNAs resulting in erroneous expression profiles. Therefore, the present review makes an attempt to highlight the various pre-analytical and analytical factors that can potentially influence the circulating miRNA levels. Literature on circulating miRNA's stability, processing and quantitation in different biofluids along with the effect of various controllable and uncontrollable factors influencing circulating miRNA expression have been summarised in the current review.
Collapse
Affiliation(s)
- Chitra Mestry
- Research Laboratories, 29537P. D. Hinduja Hospital & Medical Research Centre, Mahim, India
| | - Tester F Ashavaid
- Department of Laboratory Medicine, P. D. Hinduja Hospital & Medical Research Centre, Mahim, India
| | - Swarup Av Shah
- Department of Laboratory Medicine, P. D. Hinduja Hospital & Medical Research Centre, Mahim, India
| |
Collapse
|
|
30
|
|
|
31
|
Ren Y, Cao L, You M, Ji J, Gong Y, Ren H, Xu F, Guo H, Hu J, Li Z. “SMART” digital nucleic acid amplification technologies for lung cancer monitoring from early to advanced stages. Trends Analyt Chem 2022. [DOI: 10.1016/j.trac.2022.116774] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
|
|
32
|
Emerging digital PCR technology in precision medicine. Biosens Bioelectron 2022; 211:114344. [DOI: 10.1016/j.bios.2022.114344] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2022] [Revised: 04/23/2022] [Accepted: 05/03/2022] [Indexed: 12/20/2022]
|
|
33
|
Padinharayil H, Varghese J, John MC, Rajanikant GK, Wilson CM, Al-Yozbaki M, Renu K, Dewanjee S, Sanyal R, Dey A, Mukherjee AG, Wanjari UR, Gopalakrishnan AV, George A. Non-small cell lung carcinoma (NSCLC): Implications on molecular pathology and advances in early diagnostics and therapeutics. Genes Dis 2022. [DOI: 10.1016/j.gendis.2022.07.023] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/09/2022] Open
|
|
34
|
Divari S, Cuccato M, Fanelli A, Cannizzo FT. Development of a droplet digital PCR assay to detect illicit glucocorticoid administration in bovine. PLoS One 2022; 17:e0271613. [PMID: 35839236 PMCID: PMC9286227 DOI: 10.1371/journal.pone.0271613] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2022] [Accepted: 07/01/2022] [Indexed: 11/18/2022] Open
Abstract
Glucocorticoids are often used illegally in food-producing animals for the growth promotion of livestock animals. In accordance to official chemical methods for glucocorticoid detection, an animal is declared as non-compliant when a residue is identified in the sample. Neverthless, growth promoting molecules can often escape identification due to their rapid elimination or due to the use of non-detectable new generation drugs. Therefore, an indirect screening method able to detect the biological effect of long-term administration of low doses of dexamethasone and prednisolone on livestock has been developed to support official methods. As already described, FKBP5 (FKBP prolyl isomerase 5) expression in bovine thymus is regulated by glucocorticoids, and this specific regulation can be exploited in an indirect screening assay. In the present study, male veal calves and young bulls were considered in three different trials in which estradiol, dexamethasone, and prednisolone were administered alone or in combination with Revalor-200 subcutaneous pellets. Thoracic thymus was sampled from all animals and molecular analysis was performed. A duplex droplet digital PCR assay with EvaGreen® was employed to detect the target gene expression using absolute quantification. The developed droplet digital PCR assay was precise, showing intra- and inter-assay mean coefficient of variation values of about 6.16% and 3.17%, respectively. It was also highly specific (100%) with Youden’s index of 76.92% and 53.57% applied to veal calves and young bulls, respectively. The lowest detection limit in which the target gene expression level was kept constant, was 0.05 ng/μl of cDNA with 1 copies/μL and 0.5 copies/μL for target and reference gene, respectively. This study establishes the basis for using a digital PCR-based assay as an efficient test to identify animals illegally treated with glucocorticoids.
Collapse
Affiliation(s)
- Sara Divari
- Department of Veterinary Science, University of Turin, Grugliasco (TO), Italy
- * E-mail:
| | - Matteo Cuccato
- Department of Veterinary Science, University of Turin, Grugliasco (TO), Italy
| | - Antonella Fanelli
- Department of Veterinary Science, University of Turin, Grugliasco (TO), Italy
| | | |
Collapse
|
|
35
|
MicroRNA-Based Diagnosis and Therapy. Int J Mol Sci 2022; 23:ijms23137167. [PMID: 35806173 PMCID: PMC9266664 DOI: 10.3390/ijms23137167] [Citation(s) in RCA: 316] [Impact Index Per Article: 105.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2022] [Revised: 06/23/2022] [Accepted: 06/24/2022] [Indexed: 12/14/2022] Open
Abstract
MicroRNAs (miRNAs) are a group of endogenous non-coding RNAs that regulate gene expression. Alteration in miRNA expression results in changes in the profile of genes involving a range of biological processes, contributing to numerous human disorders. With high stability in human fluids, miRNAs in the circulation are considered as promising biomarkers for diagnosis, as well as prognosis of disease. In addition, the translation of miRNA-based therapy from a research setting to clinical application has huge potential. The aim of the current review is to: (i) discuss how miRNAs traffic intracellularly and extracellularly; (ii) emphasize the role of circulating miRNAs as attractive potential biomarkers for diagnosis and prognosis; (iii) describe how circulating microRNA can be measured, emphasizing technical problems that may influence their relative levels; (iv) highlight some of the circulating miRNA panels available for clinical use; (v) discuss how miRNAs could be utilized as novel therapeutics, and finally (v) update those miRNA-based therapeutics clinical trials that could potentially lead to a breakthrough in the treatment of different human pathologies.
Collapse
|
|
36
|
Sequeira JP, Lobo J, Constâncio V, Brito-Rocha T, Carvalho-Maia C, Braga I, Maurício J, Henrique R, Jerónimo C. DigiMir Test: Establishing a Novel Pipeline for MiR-371a Quantification Using Droplet Digital PCR in Liquid Biopsies From Testicular Germ Cell Tumor Patients. Front Oncol 2022; 12:876732. [PMID: 35756620 PMCID: PMC9226402 DOI: 10.3389/fonc.2022.876732] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2022] [Accepted: 05/03/2022] [Indexed: 01/19/2023] Open
Abstract
Testicular germ cell tumors (TGCTs) are the most common cancers in young-adult male patients aged between 15 and 39 years. Hsa-miR-371a-3p is currently the most reliable biomarker for diagnosis and monitoring of these patients non-invasively in liquid biopsies, and it is destined to be introduced in the clinic due to improved performance compared to the classical serum tumor markers available. Current studies have focused on real-time quantitative PCR (RT-qPCR) protocols for its determination; still, some challenges remain, since these protocols often require preamplification steps (costly and time-consuming), and report relative levels normalized to a housekeeping microRNA, not always performed the same way. Droplet digital PCR (ddPCR) shows the promise to overcome these challenges, skipping normalization and preamplifications, but has hardly been explored in the field of TGCTs. In this work, we provide a report of a ddPCR-based pipeline for the quantification of hsa-miR-371a-3p (the DigiMir pipeline) and compare it with two RT-qPCR protocols. A total of 107 plasma samples were investigated in the validation setting. The DigiMir pipeline detected TGCTs in a manner representative of tumor burden, with a sensitivity and specificity of 94% and 100%, respectively, outperforming the combined sensitivity of all three classical serum tumor markers (61.5%). Therefore, in this proof-of-concept investigation, we have shown that the DigiMir pipeline constitutes a new promising methodology to accurately report hsa-miR-371a-3p in the clinical setting.
Collapse
Affiliation(s)
- José Pedro Sequeira
- Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Centre (Porto.CCC), Porto, Portugal
- Master in Oncology, School of Medicine and Biomedical Sciences, University of Porto (ICBAS-UP), Porto, Portugal
| | - João Lobo
- Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Centre (Porto.CCC), Porto, Portugal
- Department of Pathology, Portuguese Oncology Institute of Porto (IPOP), Porto, Portugal
- Department of Pathology and Molecular Immunology, School of Medicine and Biomedical Sciences, University of Porto (ICBAS-UP), Porto, Portugal
| | - Vera Constâncio
- Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Centre (Porto.CCC), Porto, Portugal
- Doctoral Programme in Biomedical Sciences, School of Medicine and Biomedical Sciences, University of Porto (ICBAS-UP), Porto, Portugal
| | - Tiago Brito-Rocha
- Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Centre (Porto.CCC), Porto, Portugal
- Master in Oncology, School of Medicine and Biomedical Sciences, University of Porto (ICBAS-UP), Porto, Portugal
| | - Carina Carvalho-Maia
- Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Centre (Porto.CCC), Porto, Portugal
| | - Isaac Braga
- Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Centre (Porto.CCC), Porto, Portugal
- Department of Urology and Urology Clinics, Portuguese Oncology Institute of Porto (IPOP), Porto, Portugal
| | - Joaquina Maurício
- Department of Medical Oncology and Urology Clinics, Portuguese Oncology Institute of Porto (IPOP), Porto, Portugal
| | - Rui Henrique
- Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Centre (Porto.CCC), Porto, Portugal
- Department of Pathology, Portuguese Oncology Institute of Porto (IPOP), Porto, Portugal
- Department of Pathology and Molecular Immunology, School of Medicine and Biomedical Sciences, University of Porto (ICBAS-UP), Porto, Portugal
| | - Carmen Jerónimo
- Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Centre (Porto.CCC), Porto, Portugal
- Department of Pathology and Molecular Immunology, School of Medicine and Biomedical Sciences, University of Porto (ICBAS-UP), Porto, Portugal
| |
Collapse
|
|
37
|
Schwinghammer C, Koopmann J, Chitadze G, Karawajew L, Brüggemann M, Eckert C. Droplet Digital PCR: A New View on Minimal Residual Disease Quantification in Acute Lymphoblastic Leukemia. J Mol Diagn 2022; 24:856-866. [PMID: 35691569 DOI: 10.1016/j.jmoldx.2022.04.013] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2021] [Revised: 03/05/2022] [Accepted: 04/06/2022] [Indexed: 11/27/2022] Open
Abstract
Real-time quantitative PCR (qPCR) using immunoglobulin/T-cell receptor gene rearrangements has been used as the gold standard for minimal residual disease (MRD) monitoring in acute lymphoblastic leukemia (ALL) for >20 years. Recently, new PCR-based technologies have emerged, such as droplet digital PCR (ddPCR), which could offer several methodologic advances for MRD monitoring. In the current work, qPCR and ddPCR were compared in an unbiased blinded prospective study (n = 88 measurements) and in a retrospective study with selected critical low positive samples (n = 65 measurements). The former included flow cytometry (Flow; n = 31 measurements) as a third MRD detection method. Published guidelines (qPCR) and the latest, revised evaluation criteria (ie, ddPCR, Flow) have been applied for data analysis. The prospective study shows that ddPCR outperforms qPCR with a significantly better quantitative limit of detection and sensitivity. The number of critical MRD estimates below quantitative limit was reduced by sixfold and by threefold in the retrospective and prospective cohorts, respectively. Furthermore, the concordance of quantitative values between ddPCR and Flow was higher than between ddPCR and qPCR, probably because ddPCR and Flow are absolute quantification methods independent of the diagnostic sample, unlike qPCR. In summary, our data highlight the advantages of ddPCR as a more precise and sensitive technology that could be used to refine response monitoring in ALL.
Collapse
Affiliation(s)
- Claudia Schwinghammer
- Department of Paediatric Oncology/Haematology, Charité-Universitätsmedizin Berlin, Berlin, Germany
| | - Johannes Koopmann
- Department of Haematology, University Hospital Schleswig-Holstein, Kiel, Germany
| | - Guranda Chitadze
- Department of Haematology, University Hospital Schleswig-Holstein, Kiel, Germany
| | - Leonid Karawajew
- Department of Paediatric Oncology/Haematology, Charité-Universitätsmedizin Berlin, Berlin, Germany
| | - Monika Brüggemann
- Department of Haematology, University Hospital Schleswig-Holstein, Kiel, Germany
| | - Cornelia Eckert
- Department of Paediatric Oncology/Haematology, Charité-Universitätsmedizin Berlin, Berlin, Germany; German Cancer Consortium, German Cancer Research Center, Heidelberg, Germany.
| |
Collapse
|
|
38
|
D’Alessandra Y, Valerio V, Moschetta D, Massaiu I, Bozzi M, Conte M, Parisi V, Ciccarelli M, Leosco D, Myasoedova VA, Poggio P. Extraction-Free Absolute Quantification of Circulating miRNAs by Chip-Based Digital PCR. Biomedicines 2022; 10:biomedicines10061354. [PMID: 35740375 PMCID: PMC9220272 DOI: 10.3390/biomedicines10061354] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2022] [Revised: 06/01/2022] [Accepted: 06/03/2022] [Indexed: 11/17/2022] Open
Abstract
Circulating microRNAs (miRNA) have been proposed as specific biomarkers for several diseases. Quantitative Real-Time PCR (RT-qPCR) is the gold standard technique currently used to evaluate miRNAs expression from different sources. In the last few years, digital PCR (dPCR) emerged as a complementary and accurate detection method. When dealing with gene expression, the first and most delicate step is nucleic-acid isolation. However, all currently available protocols for RNA extraction suffer from the variable loss of RNA species due to the chemicals and number of steps involved, from sample lysis to nucleic acid elution. Here, we evaluated a new process for the detection of circulating miRNAs, consisting of sample lysis followed by direct evaluation by dPCR in plasma from healthy donors and in the cardiovascular setting. Our results showed that dPCR is able to detect, with high accuracy, low-copy-number as well as highly expressed miRNAs in human plasma samples without the need for RNA extraction. Moreover, we assessed a known myocardial infarction-related miR-133a in acute myocardial infarct patients vs. healthy subjects. In conclusion, our results show the suitability of the extraction-free quantification of circulating miRNAs as disease markers by direct dPCR.
Collapse
Affiliation(s)
- Yuri D’Alessandra
- Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy; (Y.D.); (V.V.); (D.M.); (I.M.); (M.B.); (V.A.M.)
| | - Vincenza Valerio
- Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy; (Y.D.); (V.V.); (D.M.); (I.M.); (M.B.); (V.A.M.)
| | - Donato Moschetta
- Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy; (Y.D.); (V.V.); (D.M.); (I.M.); (M.B.); (V.A.M.)
- Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, 20122 Milan, Italy
| | - Ilaria Massaiu
- Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy; (Y.D.); (V.V.); (D.M.); (I.M.); (M.B.); (V.A.M.)
| | - Michele Bozzi
- Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy; (Y.D.); (V.V.); (D.M.); (I.M.); (M.B.); (V.A.M.)
| | - Maddalena Conte
- Department of Translational Medical Sciences, University of Naples Federico II, 80138 Naples, Italy; (M.C.); (V.P.); (D.L.)
- Casa di Cura San Michele, 81024 Maddaloni, Italy
| | - Valentina Parisi
- Department of Translational Medical Sciences, University of Naples Federico II, 80138 Naples, Italy; (M.C.); (V.P.); (D.L.)
| | - Michele Ciccarelli
- Department of Medicine, Surgery and Dentistry, University of Salerno, 84084 Fisciano, Italy;
| | - Dario Leosco
- Department of Translational Medical Sciences, University of Naples Federico II, 80138 Naples, Italy; (M.C.); (V.P.); (D.L.)
| | - Veronika A. Myasoedova
- Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy; (Y.D.); (V.V.); (D.M.); (I.M.); (M.B.); (V.A.M.)
| | - Paolo Poggio
- Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy; (Y.D.); (V.V.); (D.M.); (I.M.); (M.B.); (V.A.M.)
- Correspondence: ; Tel.: +39-02-5800-2853
| |
Collapse
|
|
39
|
Mancusi A, Giordano A, Bosco A, Girardi S, Proroga YTR, Morena L, Pinto R, Sarnelli P, Cringoli G, Rinaldi L, Capuano F, Maurelli MP. Development of a droplet digital polymerase chain reaction tool for the detection of Toxoplasma gondii in meat samples. Parasitol Res 2022; 121:1467-1473. [PMID: 35230549 PMCID: PMC8993784 DOI: 10.1007/s00436-022-07477-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2021] [Accepted: 02/22/2022] [Indexed: 12/14/2022]
Abstract
Toxoplasmosis is a zoonotic disease caused by the protozoan parasite Toxoplasma gondii. Infection in humans has usually been related to the consumption of raw, undercooked or cured meat. The aim of this study was to develop a droplet digital polymerase chain reaction (ddPCR)-based assay for the detection and quantification of T. gondii in meat samples. To optimize the ddPCR, T.gondii reference DNA aliquots at five known concentrations: 8000 cg/µl, 800 cg/µl, 80 cg/µl, 8 cg/µl were used. Moreover, results obtained by ddPCR and quantitative PCR (qPCR) were compared using 80 known samples (40 positive and 40 negative), as well as 171 unknown diaphragm tissue samples collected at slaughterhouses. The ddPCR showed a sensitivity of 97.5% and a specificity of 100%, with a detection limit of 8 genomic copy/µl of T. gondii. A nearly perfect agreement (κ = 0.85) was found between results obtained by ddPCR and qPCR for both positive and negative known samples analysed. On the 171 diaphragm tissue samples from field, 7.6% resulted positive by ddPCR and only 1.2% by qPCR. Therefore, this innovative method could be very useful for the detection of T. gondii in meat samples, aiming to prevent human infections.
Collapse
Affiliation(s)
- Andrea Mancusi
- Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici, NA), Italy
| | - Angela Giordano
- Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici, NA), Italy
| | - Antonio Bosco
- Unit of Parasitology and Parasitic Diseases, Department of Veterinary Medicine and Animal Production, University of Naples Federico II, CREMOPAR, Naples, Italy
| | - Santa Girardi
- Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici, NA), Italy
| | | | - Luigi Morena
- Centro Di Riferimento Regionale Sanità Animale (CReSan), Salerno, Italy
| | - Renato Pinto
- UOD Prevenzione E Sanità Pubblica Veterinaria Regione Campania, Naples, Italy
| | - Paolo Sarnelli
- UOD Prevenzione E Sanità Pubblica Veterinaria Regione Campania, Naples, Italy
| | - Giuseppe Cringoli
- Unit of Parasitology and Parasitic Diseases, Department of Veterinary Medicine and Animal Production, University of Naples Federico II, CREMOPAR, Naples, Italy
- Centro Di Riferimento Regionale Sanità Animale (CReSan), Salerno, Italy
| | - Laura Rinaldi
- Unit of Parasitology and Parasitic Diseases, Department of Veterinary Medicine and Animal Production, University of Naples Federico II, CREMOPAR, Naples, Italy
- Centro Di Riferimento Regionale Sanità Animale (CReSan), Salerno, Italy
| | - Federico Capuano
- Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici, NA), Italy
| | - Maria Paola Maurelli
- Unit of Parasitology and Parasitic Diseases, Department of Veterinary Medicine and Animal Production, University of Naples Federico II, CREMOPAR, Naples, Italy.
| |
Collapse
|
|
40
|
Lambrescu I, Popa A, Manole E, Ceafalan LC, Gaina G. Application of Droplet Digital PCR Technology in Muscular Dystrophies Research. Int J Mol Sci 2022; 23:ijms23094802. [PMID: 35563191 PMCID: PMC9099497 DOI: 10.3390/ijms23094802] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2022] [Revised: 04/19/2022] [Accepted: 04/25/2022] [Indexed: 11/25/2022] Open
Abstract
Although they are considered rare disorders, muscular dystrophies have a strong impact on people’s health. Increased disease severity with age, frequently accompanied by the loss of ability to walk in some people, and the lack of treatment, have directed the researchers towards the development of more effective therapeutic strategies aimed to improve the quality of life and life expectancy, slow down the progression, and delay the onset or convert a severe phenotype into a milder one. Improved understanding of the complex pathology of these diseases together with the tremendous advances in molecular biology technologies has led to personalized therapeutic procedures. Different approaches that are currently under extensive investigation require more efficient, sensitive, and less invasive methods. Due to its remarkable analytical sensitivity, droplet digital PCR has become a promising tool for accurate measurement of biomarkers that monitor disease progression and quantification of various therapeutic efficiency and can be considered a tool for non-invasive prenatal diagnosis and newborn screening. Here, we summarize the recent applications of droplet digital PCR in muscular dystrophy research and discuss the factors that should be considered to get the best performance with this technology.
Collapse
Affiliation(s)
- Ioana Lambrescu
- Laboratory of Cell Biology, Neuroscience and Experimental Myology, Victor Babes National Institute of Pathology, 050096 Bucharest, Romania; (I.L.); (A.P.); (E.M.); (L.C.C.)
- Department of Cell Biology and Histology, Carol Davila University of Medicine and Pharmacy, 050474 Bucharest, Romania
| | - Alexandra Popa
- Laboratory of Cell Biology, Neuroscience and Experimental Myology, Victor Babes National Institute of Pathology, 050096 Bucharest, Romania; (I.L.); (A.P.); (E.M.); (L.C.C.)
- Department of Animal Production and Public Health, University of Agronomic Sciences and Veterinary Medicine of Bucharest, 050097 Bucharest, Romania
| | - Emilia Manole
- Laboratory of Cell Biology, Neuroscience and Experimental Myology, Victor Babes National Institute of Pathology, 050096 Bucharest, Romania; (I.L.); (A.P.); (E.M.); (L.C.C.)
- Pathology Department, Colentina Clinical Hospital, 020125 Bucharest, Romania
| | - Laura Cristina Ceafalan
- Laboratory of Cell Biology, Neuroscience and Experimental Myology, Victor Babes National Institute of Pathology, 050096 Bucharest, Romania; (I.L.); (A.P.); (E.M.); (L.C.C.)
- Department of Cell Biology and Histology, Carol Davila University of Medicine and Pharmacy, 050474 Bucharest, Romania
| | - Gisela Gaina
- Laboratory of Cell Biology, Neuroscience and Experimental Myology, Victor Babes National Institute of Pathology, 050096 Bucharest, Romania; (I.L.); (A.P.); (E.M.); (L.C.C.)
- Correspondence: ; Tel.: +40-21-319-2732
| |
Collapse
|
|
41
|
LiKidMiRs: A ddPCR-Based Panel of 4 Circulating miRNAs for Detection of Renal Cell Carcinoma. Cancers (Basel) 2022; 14:cancers14040858. [PMID: 35205607 PMCID: PMC8869982 DOI: 10.3390/cancers14040858] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2022] [Revised: 02/06/2022] [Accepted: 02/07/2022] [Indexed: 01/26/2023] Open
Abstract
Simple Summary Early detection of renal cell carcinoma (RCC) significantly increases the likelihood of curative treatment, avoiding the need of adjuvant therapies, associated side effects and comorbidities. Thus, we aimed to discover circulating microRNAs that might aid in early, minimally invasive, RCC detection/diagnosis. Abstract Background: Decreased renal cell cancer-related mortality is an important societal goal, embodied by efforts to develop effective biomarkers enabling early detection and increasing the likelihood of curative treatment. Herein, we sought to develop a new biomarker for early and minimally invasive detection of renal cell carcinoma (RCC) based on a microRNA panel assessed by ddPCR. Methods: Plasma samples from patients with RCC (n = 124) or oncocytomas (n = 15), and 64 healthy donors, were selected. Hsa-miR-21-5p, hsa-miR-126-3p, hsa-miR-155-5p and hsa-miR-200b-3p levels were evaluated using a ddPCR protocol. Results: RCC patients disclosed significantly higher circulating levels of hsa-miR-155-5p compared to healthy donors, whereas the opposite was observed for hsa-miR-21-5p levels. Furthermore, hsa-miR-21-5p and hsa-miR-155-5p panels detected RCC with high sensitivity (82.66%) and accuracy (71.89%). The hsa-miR-126-3p/hsa-miR-200b-3p panel identified the most common RCC subtype (clear cell, ccRCC) with 74.78% sensitivity. Conclusion: Variable combinations of plasma miR levels assessed by ddPCR enable accurate detection of RCC in general, and of ccRCC. These findings, if confirmed in larger studies, provide evidence for a novel ancillary tool which might aid in early detection of RCC.
Collapse
|
|
42
|
Denner J. What does the PERV copy number tell us? Xenotransplantation 2022; 29:e12732. [PMID: 35112403 DOI: 10.1111/xen.12732] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2021] [Revised: 01/05/2022] [Accepted: 01/12/2022] [Indexed: 11/27/2022]
Affiliation(s)
- Joachim Denner
- Institute of Virology, Free University Berlin, Berlin, Germany
| |
Collapse
|
|
43
|
Fan W, Wang L, Chu J, Li H, Kim EY, Cho J. Tracing Mobile DNAs: From Molecular to Population Scales. FRONTIERS IN PLANT SCIENCE 2022; 13:837378. [PMID: 35178063 PMCID: PMC8843828 DOI: 10.3389/fpls.2022.837378] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 12/16/2021] [Accepted: 01/11/2022] [Indexed: 06/14/2023]
Abstract
Transposable elements (TEs, transposons) are mobile DNAs that are prevalent in most eukaryotic genomes. In plants, their mobility has vastly contributed to genetic diversity which is essential for adaptive changes and evolution of a species. Such mobile nature of transposon has been also actively exploited in plant science research by generating genetic mutants in non-model plant systems. On the other hand, transposon mobilization can bring about detrimental effects to host genomes and they are therefore mostly silenced by the epigenetic mechanisms. TEs have been studied as major silencing targets and acted a main feature in the remarkable growth of the plant epigenetics field. Despite the importance of transposon in plant biology and biotechnology, their mobilization and the underlying mechanisms are largely left unanswered. This is mainly because of the sequence repetitiveness of transposons, which makes their detection and analyses difficult and complicated. Recently, some attempts have been made to develop new experimental methods detecting active transposons and their mobilization behavior. These techniques reveal TE mobility in various levels, including the molecular, cellular, organismal and population scales. In this review, we will highlight the novel technical approaches in the study of mobile genetic elements and discuss how these techniques impacted on the advancement of transposon research and broadened our understanding of plant genome plasticity.
Collapse
Affiliation(s)
- Wenwen Fan
- National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Ling Wang
- National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Jie Chu
- National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Hui Li
- National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Eun Yu Kim
- National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China
| | - Jungnam Cho
- National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
- CAS-JIC Centre of Excellence for Plant and Microbial Science, Chinese Academy of Sciences, Shanghai, China
| |
Collapse
|
|
44
|
Qi H, Xiong A, Jiang L, Van H, Xu J, Wu J, Zheng Q, Minervini F, Alonso DP, Yang Y, Wu L. Blood digital polymerase chain reaction as a potential method to detect human epidermal growth factor receptor 2 amplification in non-small cell lung cancer. Transl Lung Cancer Res 2022; 10:4235-4249. [PMID: 35004253 PMCID: PMC8674588 DOI: 10.21037/tlcr-21-860] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2021] [Accepted: 11/17/2021] [Indexed: 12/12/2022]
Abstract
Background This study aimed to verify the feasibility of human epidermal growth factor receptor-2 (HER2) amplification detection by digital polymerase chain reaction (dPCR) in non-small cell lung cancer (NSCLC) patients and explore whether HER2 amplification could be detected in circulating tumor DNA (ctDNA) by dPCR. Methods A total of 112 fresh biopsy tissues and 88 blood samples from NSCLC patients were collected. The serum ctDNA was obtained from blood samples. The copy number of the HER2 gene was evaluated by dPCR and next-generation sequencing (NGS). The sensitivity/specificity and survival analysis were performed by the receiver operating characteristic (ROC) curve. The survival analysis was performed by Kaplan-Meier (KM) curve and univariate Cox regression analysis was also conducted. Results ROC analysis showed a good prediction result for HER2 amplification in blood samples by dPCR. The survival analysis showed that the median overall survival (OS) in the HER2 negative group detected by blood dPCR was significantly different from the positive group. The results of multivariate Cox regression were the same as those of survival analysis. Conclusions Blood dPCR might be a potential method to detect HER2 amplification in NSCLC. Amplification of the HER2 gene detected by dPCR was correlated with OS in NSCLC.
Collapse
Affiliation(s)
- Hui Qi
- Experiment Center for Science and Technology, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Anwen Xiong
- Department of Oncology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China.,School of Medicine, Tongji University, Shanghai, China
| | - Lei Jiang
- School of Medicine, Tongji University, Shanghai, China
| | - Hardy Van
- Alphamab Oncology Ltd., Suzhou, China
| | - June Xu
- Alphamab Oncology Ltd., Suzhou, China
| | - Jing Wu
- Genetron Health (Beijing) Co. Ltd., Beijing, China
| | | | - Fabrizio Minervini
- Department of Thoracic Surgery, Cantonal Hospital Lucerne, Lucerne, Switzerland
| | - Dinora Polanco Alonso
- Group of Translational Research in Respiratory Medicine, Hospital Universitari Arnau de Vilanova y Santa Maria, IRB Lleida, Lleida, Spain
| | - Yifu Yang
- Experiment Center for Science and Technology, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Liang Wu
- Department of Thoracic Surgery, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| |
Collapse
|
|
45
|
Zhang Z, Li X, Liu H, Zamyadi A, Guo W, Wen H, Gao L, Nghiem LD, Wang Q. Advancements in detection and removal of antibiotic resistance genes in sludge digestion: A state-of-art review. BIORESOURCE TECHNOLOGY 2022; 344:126197. [PMID: 34710608 DOI: 10.1016/j.biortech.2021.126197] [Citation(s) in RCA: 33] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/05/2021] [Revised: 10/16/2021] [Accepted: 10/19/2021] [Indexed: 06/13/2023]
Abstract
Sludge from wastewater treatment plants can act as a repository and crucial environmental provider of antibiotic resistance genes (ARGs). Over the past few years, people's knowledge regarding the occurrence and removal of ARGs in sludge has broadened remarkably with advancements in molecular biological techniques. Anaerobic and aerobic digestion were found to effectively achieve sludge reduction and ARGs removal. This review summarized advanced detection and removal techniques of ARGs, in the last decade, in the sludge digestion field. The fate of ARGs due to different sludge digestion strategies (i.e., anaerobic and aerobic digestion under mesophilic or thermophilic conditions, and in combination with relevant pretreatment technologies (e.g., thermal hydrolysis pretreatment, microwave pretreatment and alkaline pretreatment) and additives (e.g., ferric chloride and zero-valent iron) were systematically summarized and compared in this review. To date, this is the first review that provides a comprehensive assessment of the state-of-the-art technologies and future recommendations.
Collapse
Affiliation(s)
- Zehao Zhang
- Centre for Technology in Water and Wastewater, School of Civil and Environmental Engineering, University of Technology Sydney, Ultimo, NSW 2007, Australia
| | - Xuan Li
- School of Civil, Mining and Environmental Engineering, University of Wollongong, Australia.
| | - Huan Liu
- Centre for Technology in Water and Wastewater, School of Civil and Environmental Engineering, University of Technology Sydney, Ultimo, NSW 2007, Australia
| | - Arash Zamyadi
- Water Research Australia Limited, Melbourne & Adelaide SA 5001, Australia
| | - Wenshan Guo
- Centre for Technology in Water and Wastewater, School of Civil and Environmental Engineering, University of Technology Sydney, Ultimo, NSW 2007, Australia
| | - Haiting Wen
- School of Environment and Nature Resources, Renmin University of China, Beijing 100872, PR China
| | - Li Gao
- South East Water, 101 Wells Street, Frankston, VIC 3199, Australia
| | - Long D Nghiem
- Centre for Technology in Water and Wastewater, School of Civil and Environmental Engineering, University of Technology Sydney, Ultimo, NSW 2007, Australia
| | - Qilin Wang
- Centre for Technology in Water and Wastewater, School of Civil and Environmental Engineering, University of Technology Sydney, Ultimo, NSW 2007, Australia
| |
Collapse
|
|
46
|
Circulating microRNA-197-3p as a potential biomarker for asbestos exposure. Sci Rep 2021; 11:23955. [PMID: 34907223 PMCID: PMC8671556 DOI: 10.1038/s41598-021-03189-9] [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] [Received: 08/31/2021] [Accepted: 11/24/2021] [Indexed: 12/15/2022] Open
Abstract
Asbestos is considered the main cause of diseases in workers exposed to this mineral in the workplace as well as an environmental pollutant. The association between asbestos and the onset of different diseases has been reported, but asbestos exposure specific biomarkers are not known. MicroRNAs (miRNAs) are small, single-strand, non-coding RNAs, with potential value as diagnostic, prognostic, and predictive markers in liquid biopsies. Sera collected from workers ex-exposed to asbestos (WEA) fibers were compared with sera from healthy subjects (HS) of similar age, as liquid biopsies. The expression of the circulating miRNA 197-3p was investigated employing two different highly analytical PCR methods, i.e. RT-qPCR and ddPCR. MiR-197-3p levels were tested in sera from WEA compared to HS. MiR-197-3p tested dysregulated in sera from WEA (n = 75) compared to HS (n = 62). Indeed, miR-197-3p was found to be 2.6 times down-regulated in WEA vs. HS (p = 0.0001***). In addition, an inverse correlation was detected between miR-197-3p expression level and cumulative asbestos exposure, being this miRNA down-regulated 2.1 times in WEA, with high cumulative asbestos exposure, compared to WEA with low exposure (p = 0.0303*). Circulating miR-197-3p, found to be down regulated in sera from WEA, is proposed as a new potential biomarker of asbestos exposure.
Collapse
|
|
47
|
Schofield AL, Brown JP, Brown J, Wilczynska A, Bell C, Glaab WE, Hackl M, Howell L, Lee S, Dear JW, Remes M, Reeves P, Zhang E, Allmer J, Norris A, Falciani F, Takeshita LY, Seyed Forootan S, Sutton R, Park BK, Goldring C. Systems analysis of miRNA biomarkers to inform drug safety. Arch Toxicol 2021; 95:3475-3495. [PMID: 34510227 PMCID: PMC8492583 DOI: 10.1007/s00204-021-03150-9] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2021] [Accepted: 08/23/2021] [Indexed: 02/06/2023]
Abstract
microRNAs (miRNAs or miRs) are short non-coding RNA molecules which have been shown to be dysregulated and released into the extracellular milieu as a result of many drug and non-drug-induced pathologies in different organ systems. Consequently, circulating miRs have been proposed as useful biomarkers of many disease states, including drug-induced tissue injury. miRs have shown potential to support or even replace the existing traditional biomarkers of drug-induced toxicity in terms of sensitivity and specificity, and there is some evidence for their improved diagnostic and prognostic value. However, several pre-analytical and analytical challenges, mainly associated with assay standardization, require solutions before circulating miRs can be successfully translated into the clinic. This review will consider the value and potential for the use of circulating miRs in drug-safety assessment and describe a systems approach to the analysis of the miRNAome in the discovery setting, as well as highlighting standardization issues that at this stage prevent their clinical use as biomarkers. Highlighting these challenges will hopefully drive future research into finding appropriate solutions, and eventually circulating miRs may be translated to the clinic where their undoubted biomarker potential can be used to benefit patients in rapid, easy to use, point-of-care test systems.
Collapse
Affiliation(s)
- Amy L Schofield
- MRC Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, University of Liverpool, Sherrington Buildings, Ashton Street, Liverpool, L69 3GE, UK
| | - Joseph P Brown
- MRC Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, University of Liverpool, Sherrington Buildings, Ashton Street, Liverpool, L69 3GE, UK
| | - Jack Brown
- MRC Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, University of Liverpool, Sherrington Buildings, Ashton Street, Liverpool, L69 3GE, UK
| | - Ania Wilczynska
- bit.bio, Babraham Research Campus, The Dorothy Hodgkin Building, Cambridge, CB22 3FH, UK
| | - Catherine Bell
- CVRM Safety, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Gothenburg, Sweden
| | - Warren E Glaab
- Merck & Co., Inc, 770 Sumneytown Pike, West Point, PA, 19486, USA
| | | | - Lawrence Howell
- GlaxoSmithKline (GSK), Stevenage, Greater Cambridge Area, UK
| | - Stephen Lee
- ABHI, 1 Duchess St, 4th Floor, Suite 2, London, W1W 6AN, UK
| | - James W Dear
- Centre for Cardiovascular Science, The Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK
| | - Mika Remes
- Genomics EMEA, QIAGEN Aarhus, Prismet, Silkeborgvej 2, 8000, Aarhus C, Denmark
| | - Paul Reeves
- Arcis Biotechnology Limited, Suite S07, Techspace One, Sci-tech Daresbury, Keckwick Lane, Daresbury, Warrington, WA4 4AB, UK
| | - Eunice Zhang
- Wolfson Centre for Personalised Medicine, Department of Pharmacology and Therapeutics, University of Liverpool, Crown Street, Liverpool, L69 3BX, UK
| | - Jens Allmer
- Applied Bioinformatics, Bioscience, Wageningen University and Research, Droevendaalsesteeg 4, 6708 PB, Wageningen, The Netherlands
| | - Alan Norris
- MRC Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, University of Liverpool, Sherrington Buildings, Ashton Street, Liverpool, L69 3GE, UK
| | - Francesco Falciani
- Computational Biology Facility, MerseyBio, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK
| | - Louise Y Takeshita
- Computational Biology Facility, MerseyBio, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK
| | - Shiva Seyed Forootan
- MRC Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, University of Liverpool, Sherrington Buildings, Ashton Street, Liverpool, L69 3GE, UK
| | - Robert Sutton
- Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Biosciences Building, Crown Street, Liverpool, L69 7BE, UK
| | - B Kevin Park
- MRC Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, University of Liverpool, Sherrington Buildings, Ashton Street, Liverpool, L69 3GE, UK
| | - Chris Goldring
- MRC Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, University of Liverpool, Sherrington Buildings, Ashton Street, Liverpool, L69 3GE, UK.
| |
Collapse
|
|
48
|
Unveiling the World of Circulating and Exosomal microRNAs in Renal Cell Carcinoma. Cancers (Basel) 2021; 13:cancers13215252. [PMID: 34771419 PMCID: PMC8582552 DOI: 10.3390/cancers13215252] [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: 09/23/2021] [Accepted: 10/18/2021] [Indexed: 02/07/2023] Open
Abstract
Simple Summary Liquid biopsies have emerged as a new tool for early diagnosis. In renal cell carcinoma, this need is also evident and may represent an improvement in disease management. Hence, in this review we discuss the most updated advances in the assessment of miRNAs in liquid biopsies. Moreover, we explore the potential of circulating or exosome miRNAs in renal cell carcinoma to overcome the tissue biopsies limitations. Abstract Renal cell carcinoma is the third most common urological cancer. Despite recent advances, late diagnosis and poor prognosis of advanced-stage disease remain a major problem, entailing the need for novel early diagnosis tools. Liquid biopsies represent a promising minimally invasive clinical tool, providing real-time feedback of tumor behavior and biological potential, addressing its clonal evolution and representing its heterogeneity. In particular, the study of circulating microRNAs and exosomal microRNAs in liquid biopsies experienced an exponential increase in recent years, considering the potential clinical utility and available technology that facilitates implementation. Herein, we provide a systematic review on the applicability of these biomarkers in the context of renal cell carcinoma. Issues such as additional benefit from extracting microRNAs transported in extracellular vesicles, use for subtyping and representation of different histological types, correlation with tumor burden, and prediction of patient outcome are also addressed. Despite the need for more conclusive research, available data indicate that exosomal microRNAs represent a robust minimally invasive biomarker for renal cell carcinoma. Thus, innovative research on microRNAs and novel detection techniques are likely to provide clinically relevant biomarkers, overcome current clinical challenges, and improve patient management.
Collapse
|
|
49
|
Antonello M, Scutari R, Lauricella C, Renica S, Motta V, Torri S, Russo C, Gentile L, Cento V, Colagrossi L, Mattana G, Codecasa LR, Vismara C, Scaglione F, Veronese SM, Bonoldi E, Bandera A, Gori A, Mazzola E, Perno CF, Alteri C. Rapid Detection and Quantification of Mycobacterium tuberculosis DNA in Paraffinized Samples by Droplet Digital PCR: A Preliminary Study. Front Microbiol 2021; 12:727774. [PMID: 34589075 PMCID: PMC8475183 DOI: 10.3389/fmicb.2021.727774] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2021] [Accepted: 08/16/2021] [Indexed: 01/23/2023] Open
Abstract
Background: Rapid and reliable diagnosis of tuberculosis (TB) represents a diagnostic challenge in compartmentalized extrapulmonary TB infection because of the small number of mycobacteria (MTB) and the frequent lack of fresh samples to perform culture. Here, we estimate the performances of homemade droplet digital PCR (ddPCR)-based assays against culture in 89 biopsies, for those fresh and formalin-fixed and paraffin-embedded (FFPE) subsamples were available. Methods: MTB diagnosis in fresh subsamples was performed by culture. Fresh subsamples were also analyzed for acid-fast bacilli smear-microscopy (AFB) and Xpert® MTB/RIF (Xpert). MTB examination was repeated in blind in the 89 FFPE subsamples by in-house ddPCR assays targeting the IS6110 and rpoB. Analytical sensitivity of ddPCR assays was evaluated using serial dilution of H37Rv strain. Limit of detection (LOD) was calculated by probit analysis. Results were expressed in copies/106 cells. Results: IS6110 and rpoB ddPCR assays showed a good linear correlation between expected and observed values (R2: 0.9907 and 0.9743, respectively). Probit analyses predicted a LOD of 17 and 40 copies/106 cells of MTB DNA for IS6110 and rpoB, respectively. Of the 89 biopsies, 68 were culture positive and 21 were culture negative. Considering mycobacterial culture as reference method, IS6110 assay yielded positive results in 67/68 culture-positive samples with a median interquartile range (IQR) of 1,680 (550–8,444) copies/106 cells (sensitivity: 98.5%; accuracy: 98.9). These performances were superior to those reported by the rpoB assay in FFPE subsamples (sensitivity: 66.20%; accuracy: 74.1) and even superior to those reported by Xpert and AFB in fresh subsamples (sensitivity: 79.4 and 33.8%, respectively; accuracy: 84.3 and 49.4, respectively). When Xpert and AFB results were stratified according to mycobacterial load detected by rpoB and IS6110 ddPCR, bacterial load was lower in Xpert and AFB negative with respect to Xpert and AFB-positive samples (p = 0.003 and 0.01 for rpoB and p = 0.01 and 0.11 for IS6110), confirming the poor sensitivity of these methods in paucibacillary disease. Conclusion: ddPCR provides highly sensitive, accurate, and rapid MTB diagnosis in FFPE samples, as defined by the high concordance between IS6110 assay and culture results. This approach can be safely introduced in clinical routine to accelerate MTB diagnosis mainly when culture results remain unavailable.
Collapse
Affiliation(s)
- Maria Antonello
- Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy
| | - Rossana Scutari
- Department of Experimental Medicine, University of Rome "Tor Vergata,"Rome, Italy
| | - Calogero Lauricella
- Department of Pathology, ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy
| | - Silvia Renica
- Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy
| | - Valentina Motta
- Department of Pathology, ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy
| | - Stefania Torri
- Unit of Microbiology, Department of Chemical-Clinical and Microbiology Analyses, ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy
| | - Cristina Russo
- Unit of Microbiology and Diagnostic Immunology, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
| | - Leonarda Gentile
- Unit of Microbiology and Diagnostic Immunology, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
| | - Valeria Cento
- Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy
| | - Luna Colagrossi
- Unit of Microbiology and Diagnostic Immunology, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
| | - Giordana Mattana
- Unit of Microbiology and Diagnostic Immunology, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
| | - Luigi Ruffo Codecasa
- Regional TB Reference Centre, Villa Marelli Institute, ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy
| | - Chiara Vismara
- Unit of Microbiology, Department of Chemical-Clinical and Microbiology Analyses, ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy
| | - Francesco Scaglione
- Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy.,Unit of Microbiology, Department of Chemical-Clinical and Microbiology Analyses, ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy
| | | | - Emanuela Bonoldi
- Department of Pathology, ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy
| | - Alessandra Bandera
- Infectious Disease Unit, Department of Internal Medicine, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milan, Italy.,Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy
| | - Andrea Gori
- Infectious Disease Unit, Department of Internal Medicine, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milan, Italy.,Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy
| | - Ester Mazzola
- Unit of Microbiology, Department of Chemical-Clinical and Microbiology Analyses, ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy
| | - Carlo Federico Perno
- Unit of Microbiology and Diagnostic Immunology, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.,Multimodal Medicine Research Area, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
| | - Claudia Alteri
- Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy.,Multimodal Medicine Research Area, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
| |
Collapse
|
|
50
|
Emerging methods for and novel insights gained by absolute quantification of mitochondrial DNA copy number and its clinical applications. Pharmacol Ther 2021; 232:107995. [PMID: 34592204 DOI: 10.1016/j.pharmthera.2021.107995] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2021] [Revised: 08/26/2021] [Accepted: 09/01/2021] [Indexed: 02/07/2023]
Abstract
The past thirty years have seen a surge in interest in pathophysiological roles of mitochondria, and the accurate quantification of mitochondrial DNA copy number (mCN) in cells and tissue samples is a fundamental aspect of assessing changes in mitochondrial health and biogenesis. Quantification of mCN between studies is surprisingly variable due to a combination of physiological variability and diverse protocols being used to measure this endpoint. The advent of novel methods to quantify nucleic acids like digital polymerase chain reaction (dPCR) and high throughput sequencing offer the ability to measure absolute values of mCN. We conducted an in-depth survey of articles published between 1969 -- 2020 to create an overview of mCN values, to assess consensus values of tissue-specific mCN, and to evaluate consistency between methods of assessing mCN. We identify best practices for methods used to assess mCN, and we address the impact of using specific loci on the mitochondrial genome to determine mCN. Current data suggest that clinical measurement of mCN can provide diagnostic and prognostic value in a range of diseases and health conditions, with emphasis on cancer and cardiovascular disease, and the advent of means to measure absolute mCN should improve future clinical applications of mCN measurements.
Collapse
|