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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.

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