Evaluation and validation of suitable reference genes for reverse transcription-quantitative polymerase chain reaction studies in cholangiocarcinoma patients and cell lines

Yang,Jinghui; Yang,Qiwei; Yu,Shan; Zhang,Xuewen

doi:10.3892/ol.2016.4232

Evaluation and validation of suitable reference genes for reverse transcription-quantitative polymerase chain reaction studies in cholangiocarcinoma patients and cell lines

Authors:
- Jinghui Yang
- Qiwei Yang
- Shan Yu
- Xuewen Zhang
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Affiliations: Department of Hepatopancreatobiliary Surgery, China‑Japan Union Hospital of Jilin University, Changchun, Jilin 130033, P.R. China, Central Laboratory, Second Hospital of Jilin University, Changchun, Jilin 130041, P.R. China, Department of Neurology, China‑Japan Union Hospital of Jilin University, Changchun, Jilin 130033, P.R. China
Published online on: February 17, 2016 https://doi.org/10.3892/ol.2016.4232
Pages: 2673-2681

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Abstract

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has become a frequently used method in gene expression studies. The relative quantification method is an important and common method for the evaluation of RT-qPCR data. One of the key requirements of this method is to identify an applicable internal reference gene. However, to the best of our knowledge, no suitable reference genes have been identified for the genetic analysis of cholangiocarcinoma (CCA) in humans and cell lines. In the present study, screening was conducted using 12 common reference genes, which were selected in order to provide an experimental basis for the study of the gene expression in CCA patients and cell lines. Tumor samples and adjacent non‑neoplastic tissues from 20 patients with CCA were obtained for the present study. The stability and applicability of the 12 reference gene candidates were validated using GeNorm, NormFinder and BestKeeper software. Based on a comparison of the results, the following reference genes are recommended for various tissue groups: Total sample group, ribosomal protein L29; CCA tissue group, TATA‑box binding protein; CCA cell line and tissue group, actin‑β; CCA cell line group, 18S ribosomal RNA; CCA tissue and adjacent non‑neoplastic tissue group, 5'‑aminolevulinate synthase 1; and adjacent non‑neoplastic tissue group, hypoxanthine phosphoribosyltransferase 1. The results of the search also clearly reveal that a systematic study regarding the selection of suitable reference genes for studying the target gene profiling in CCA tissues and cell lines has not been previously published. The present study may provide useful information for future studies that examine the gene expression of CCA for choosing suitable reference genes.

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