Format

Send to

Choose Destination
Sci Rep. 2018 Jan 31;8(1):1949. doi: 10.1038/s41598-018-19458-z.

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

Zhao H1,2, Ma TF1,3, Lin J3, Liu LL1,3, Sun WJ1,3, Guo LX1,3, Wang SQ1,3, Otecko NO4,5, Zhang YP6,7.

Author information

1
State Key Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming, China.
2
Key Laboratory for Animal Genetic Diversity and Evolution of High Education in Yunnan Province, Yunnan University, Kunming, Yunnan, China.
3
School of Life Science, Yunnan University, Kunming, 650091, China.
4
State Key Laboratory of Genetic Resources and Evolution & Yunnan Laboratory of Molecular Biology of Domestic Animals & Germplasm Bank of Wild Species, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650223, China.
5
Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, 650204, China.
6
State Key Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming, China. zhangyp@mail.kiz.ac.cn.
7
State Key Laboratory of Genetic Resources and Evolution & Yunnan Laboratory of Molecular Biology of Domestic Animals & Germplasm Bank of Wild Species, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650223, China. zhangyp@mail.kiz.ac.cn.

Abstract

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.

PMID:
29386530
PMCID:
PMC5792445
DOI:
10.1038/s41598-018-19458-z
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Nature Publishing Group Icon for PubMed Central
Loading ...
Support Center