Identification of valid reference genes for mRNA and microRNA normalisation in prostate cancer cell lines

RT-qPCR offers high sensitivity, for accurate interpretations of qPCR results however, normalisation using suitable reference genes is fundamental. Androgens can regulate transcriptional expression including reference gene expression in prostate cancer. In this study, we evaluated ten mRNA and six n...

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Autores principales: Zhao, Hui, Ma, Teng-Fei, Lin, Jie, Liu, Lin-Lin, Sun, Wei-Jie, Guo, Li-Xia, Wang, Si-Qi, Otecko, Newton O., Zhang, Ya-Ping
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
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Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792445/
https://www.ncbi.nlm.nih.gov/pubmed/29386530
http://dx.doi.org/10.1038/s41598-018-19458-z
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author Zhao, Hui
Ma, Teng-Fei
Lin, Jie
Liu, Lin-Lin
Sun, Wei-Jie
Guo, Li-Xia
Wang, Si-Qi
Otecko, Newton O.
Zhang, Ya-Ping
author_facet Zhao, Hui
Ma, Teng-Fei
Lin, Jie
Liu, Lin-Lin
Sun, Wei-Jie
Guo, Li-Xia
Wang, Si-Qi
Otecko, Newton O.
Zhang, Ya-Ping
author_sort Zhao, Hui
collection PubMed
description RT-qPCR offers high sensitivity, for accurate interpretations of qPCR results however, normalisation using suitable reference genes is fundamental. Androgens can regulate transcriptional expression including reference gene expression in prostate cancer. In this study, we evaluated ten mRNA and six non-protein coding RNA reference genes in five prostate cell lines under varied dihydrotestosterone (DHT) treatments. We validated the effects of DHT-treatments using media containing charcoal-stripped serum prior to DHT stimulation on the test samples by Western blot experiments. Reference gene expression stability was analysed using three programs (geNorm, NormFinder and BestKeeper), and the recommended comprehensive ranking is provided. Our results reveal that ACTB and GAPDH, and miR-16 and miR-1228-3p are the most suitable mRNA and miRNA reference genes across all cell lines, respectively. Considering prostate cancer cell types, ACTB/GAPDH and ACTB/HPRT1 are the most suitable reference gene combinations for mRNA analysis, and miR-16/miR-1228-3p and RNU6-2/RNU43 for miRNA analysis in AR+, and AR− and normal cell lines, respectively. Comparison of relative target gene (PCA3 and miR-141) expression reveals different patterns depending on reference genes used for normalisation. To our knowledge, this is the first report on validation of reference genes under different DHT treatments in prostate cancer cells. This study provides insights for discovery of reliable DHT-regulated genes in prostate cells.
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spelling pubmed-57924452018-02-12 Identification of valid reference genes for mRNA and microRNA normalisation in prostate cancer cell lines Zhao, Hui Ma, Teng-Fei Lin, Jie Liu, Lin-Lin Sun, Wei-Jie Guo, Li-Xia Wang, Si-Qi Otecko, Newton O. Zhang, Ya-Ping Sci Rep Article RT-qPCR offers high sensitivity, for accurate interpretations of qPCR results however, normalisation using suitable reference genes is fundamental. Androgens can regulate transcriptional expression including reference gene expression in prostate cancer. In this study, we evaluated ten mRNA and six non-protein coding RNA reference genes in five prostate cell lines under varied dihydrotestosterone (DHT) treatments. We validated the effects of DHT-treatments using media containing charcoal-stripped serum prior to DHT stimulation on the test samples by Western blot experiments. Reference gene expression stability was analysed using three programs (geNorm, NormFinder and BestKeeper), and the recommended comprehensive ranking is provided. Our results reveal that ACTB and GAPDH, and miR-16 and miR-1228-3p are the most suitable mRNA and miRNA reference genes across all cell lines, respectively. Considering prostate cancer cell types, ACTB/GAPDH and ACTB/HPRT1 are the most suitable reference gene combinations for mRNA analysis, and miR-16/miR-1228-3p and RNU6-2/RNU43 for miRNA analysis in AR+, and AR− and normal cell lines, respectively. Comparison of relative target gene (PCA3 and miR-141) expression reveals different patterns depending on reference genes used for normalisation. To our knowledge, this is the first report on validation of reference genes under different DHT treatments in prostate cancer cells. This study provides insights for discovery of reliable DHT-regulated genes in prostate cells. Nature Publishing Group UK 2018-01-31 /pmc/articles/PMC5792445/ /pubmed/29386530 http://dx.doi.org/10.1038/s41598-018-19458-z Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Zhao, Hui
Ma, Teng-Fei
Lin, Jie
Liu, Lin-Lin
Sun, Wei-Jie
Guo, Li-Xia
Wang, Si-Qi
Otecko, Newton O.
Zhang, Ya-Ping
Identification of valid reference genes for mRNA and microRNA normalisation in prostate cancer cell lines
title Identification of valid reference genes for mRNA and microRNA normalisation in prostate cancer cell lines
title_full Identification of valid reference genes for mRNA and microRNA normalisation in prostate cancer cell lines
title_fullStr Identification of valid reference genes for mRNA and microRNA normalisation in prostate cancer cell lines
title_full_unstemmed Identification of valid reference genes for mRNA and microRNA normalisation in prostate cancer cell lines
title_short Identification of valid reference genes for mRNA and microRNA normalisation in prostate cancer cell lines
title_sort identification of valid reference genes for mrna and microrna normalisation in prostate cancer cell lines
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792445/
https://www.ncbi.nlm.nih.gov/pubmed/29386530
http://dx.doi.org/10.1038/s41598-018-19458-z
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