Brief Article
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World J Gastroenterol. Nov 7, 2013; 19(41): 7121-7128
Published online Nov 7, 2013. doi: 10.3748/wjg.v19.i41.7121
Reference genes for quantitative RT-PCR data in gastric tissues and cell lines
Fernanda Wisnieski, Danielle Queiroz Calcagno, Mariana Ferreira Leal, Leonardo Caires dos Santos, Carolina de Oliveira Gigek, Elizabeth Suchi Chen, Thaís Brilhante Pontes, Paulo Pimentel Assumpção, Mônica Barauna de Assumpção, Sâmia Demachki, Rommel Rodríguez Burbano, Marília de Arruda Cardoso Smith
Fernanda Wisnieski, Danielle Queiroz Calcagno, Mariana Ferreira Leal, Leonardo Caires dos Santos, Carolina de Oliveira Gigek, Elizabeth Suchi Chen, Thaís Brilhante Pontes, Marília de Arruda Cardoso Smith, Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de São Paulo, São Paulo 04023900, Brazil
Mariana Ferreira Leal, Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo 04038031, Brazil
Paulo Pimentel Assumpção, Mônica Barauna de Assumpção, Sâmia Demachki, Núcleo de Pesquisa em Oncologia, Hospital João de Barros Barreto, Universidade Federal do Pará, Belém 60673000, Brazil
Rommel Rodríguez Burbano, Laboratório de Citogenética Humana, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém 66075110, Brazil
Author contributions: Smith MAC, Burbano RR, Calcagno DQ and Wisnieski F designed the research; Wisnieski F, Calcagno DQ, Santos LC, Gigek CO, Pontes TB, Assumpção PP, Assumpção MB and Demachki S performed the research; Wisnieski F, Leal MF and Chen ES analyzed the data; Smith MAC, Burbano RR, Wisnieski F, Leal MF and Calcagno DQ wrote the paper.
Supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) to Wisnieski F and Gigek CO; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) to Burbano RR and Smith MAC; and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) to Wisnieski F, Calcagno DQ, Leal M, Pontes TB and Smith MAC as grants and fellowship awards
Correspondence to: Fernanda Wisnieski, PhD, Genetics Division, Department of Morphology and Genetic, Federal University of São Paulo, São Paulo 04023900, Brazil. fernandawis@yahoo.com.br
Telephone: +55-11-55764260 Fax: +55-11-55764264
Received: February 7, 2013
Revised: August 6, 2013
Accepted: August 20, 2013
Published online: November 7, 2013
Processing time: 284 Days and 13.9 Hours
Abstract

AIM: To evaluate the suitability of reference genes in gastric tissue samples and cell lines.

METHODS: The suitability of genes ACTB, B2M, GAPDH, RPL29, and 18S rRNA was assessed in 21 matched pairs of neoplastic and adjacent non-neoplastic gastric tissues from patients with gastric adenocarcinoma, 27 normal gastric tissues from patients without cancer, and 4 cell lines using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The ranking of the best single and combination of reference genes was determined by NormFinder, geNorm™, BestKeeper, and DataAssist™. In addition, GenEx software was used to determine the optimal number of reference genes. To validate the results, the mRNA expression of a target gene, DNMT1, was quantified using the different reference gene combinations suggested by the various software packages for normalization.

RESULTS: ACTB was the best reference gene for all gastric tissues, cell lines and all gastric tissues plus cell lines. GAPDH + B2M or ACTB + B2M was the best combination of reference genes for all the gastric tissues. On the other hand, ACTB + B2M was the best combination for all the cell lines tested and was also the best combination for analyses involving all the gastric tissues plus cell lines. According to the GenEx software, 2 or 3 genes were the optimal number of references genes for all the gastric tissues. The relative quantification of DNMT1 showed similar patterns when normalized by each combination of reference genes. The level of expression of DNMT1 in neoplastic, adjacent non-neoplastic and normal gastric tissues did not differ when these samples were normalized using GAPDH + B2M (P = 0.32), ACTB + B2M (P = 0.61), or GAPDH + B2M + ACTB (P = 0.44).

CONCLUSION: GAPDH + B2M or ACTB + B2M is the best combination of reference gene for all the gastric tissues, and ACTB + B2M is the best combination for the cell lines tested.

Keywords: Gastric cancer; Reference gene; Normalization; Gene expression; Quantitative real-time polymerase chain reaction

Core tip: Gene expression studies have revealed much about the molecular basis of gastric cancer. However, the normalization of expression data using reference genes without validation may undermine the results. In the present study, we evaluated the suitability of possible reference genes in gastric tissues and cell lines. To our knowledge, our study is the first to determine and validate reference genes for gastric samples in a Western population. In addition, the inclusion of normal gastric tissues from patients without cancer in determining the best reference genes is original in the literature.