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Validation of reference genes for the normalization of RT-qPCR expression studies in human tongue carcinoma cell lines and tissue.

ABSTRACT: Reverse transcription quantitative polymerase chain reaction (RT-qPCR) has become a frequently used strategy in gene expression studies. The relative quantification method is an important and commonly used method for the evaluation of RT-qPCR data. The key aim of this method is to identify an applicable internal reference gene. However, there are currently no data concerning the expression of reference genes for gene analysis in human tongue carcinoma cell lines and tissues. In the present study, screening was performed using 12 common reference genes, which were selected in order to provide an experimental basis for the investigation of gene expression in human tongue carcinoma. Tca-8113 and CAL-27 cell lines and a total of 8 tongue carcinoma tissue samples were investigated. The gene expression stability and the applicability of the 12 reference gene candidates were determined using the geNorm, NormFinder and BestKeeper software programs. The results from the three software programs were demonstrated to be variable following comparison. The recommended combinations were 5'-aminolevulinate synthase 1 + glucuronidase ? + ribosomal protein L29 (RPL29) for the cell line + tissue group, ?-2-microglobulin + RPL29 for the cell line group and peptidylprolyl isomerase A + hydroxymethylbilane synthase + RPL29 for the tissue group. These recommended internal reference genes may improve the accuracy of relative quantitation analysis of target gene expression performed by the RT-qPCR method in further gene expression research on human tongue carcinoma.

SUBMITTER: Wang X

PROVIDER: S-EPMC5431254 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Validation of reference genes for the normalization of RT-qPCR expression studies in human tongue carcinoma cell lines and tissue.

Wang Xiaofeng X Liu Xin X Liu Cong C Ren Ming M Gao Sujie S Zhao Guanjie G Zhang Tianfu T Yang Qiwei Q

Oncology letters 20170322 5

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) has become a frequently used strategy in gene expression studies. The relative quantification method is an important and commonly used method for the evaluation of RT-qPCR data. The key aim of this method is to identify an applicable internal reference gene. However, there are currently no data concerning the expression of reference genes for gene analysis in human tongue carcinoma cell lines and tissues. In the present study ...[more]

PMID: 28521492

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Project description:A critical step in the RT-qPCR workflow for studying gene expression is data normalization, one of the strategies being the use of reference genes. This study aimed to identify and validate a selection of reference genes for relative quantification in Talaromyces versatilis, a relevant industrial filamentous fungus. Beyond T. versatilis, this study also aimed to propose reference genes that are applicable more widely for RT-qPCR data normalization in filamentous fungi.A selection of stable, potential reference genes was carried out in silico from RNA-seq based transcriptomic data obtained from T. versatilis. A dozen functionally unrelated candidate genes were analysed by RT-qPCR assays over more than 30 relevant culture conditions. By using geNorm, we showed that most of these candidate genes had stable transcript levels in most of the conditions, from growth environments to conidial germination. The overall robustness of these genes was explored further by showing that any combination of 3 of them led to minimal normalization bias. To extend the relevance of the study beyond T. versatilis, we challenged their stability together with sixteen other classically used genes such as ?-tubulin or actin, in a representative sample of about 100 RNA-seq datasets. These datasets were obtained from 18 phylogenetically distant filamentous fungi exposed to prevalent experimental conditions. Although this wide analysis demonstrated that each of the chosen genes exhibited sporadic up- or down-regulation, their hierarchical clustering allowed the identification of a promising group of 6 genes, which presented weak expression changes and no tendency to up- or down-regulation over the whole set of conditions. This group included ubcB, sac7, fis1 and sarA genes, as well as TFC1 and UBC6 that were previously validated for their use in S. cerevisiae.We propose a set of 6 genes that can be used as reference genes in RT-qPCR data normalization in any field of fungal biology. However, we recommend that the uniform transcription of these genes is tested by systematic experimental validation and to use the geometric averaging of at least 3 of the best ones. This will minimize the bias in normalization and will support trustworthy biological conclusions.

Dataset Information

Validation of reference genes for the normalization of RT-qPCR expression studies in human tongue carcinoma cell lines and tissue.

Publications

Validation of reference genes for the normalization of RT-qPCR expression studies in human tongue carcinoma cell lines and tissue.

Similar Datasets

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