Validation of suitable normalizers for miR expression patterns analysis covering tumour heterogeneity

Oncogenic microRNAs (miRs) have emerged as diagnostic biomarkers and novel molecular targets for anti-cancer drug therapies. Real-time quantitative PCR (qPCR) is one of the most powerful techniques for analyzing miRs; however, the use of unsuitable normalizers might bias the results. Tumour heterogeneity makes even more difficult the selection of an adequate endogenous normalizer control. Here, we have evaluated five potential referenced small RNAs (U6, rRNA5s, SNORD44, SNORD24 and hsa-miR-24c-3p) using RedFinder algorisms to perform a stability expression analysis in i) normal colon cells, ii) colon and breast cancer cell lines and iii) cancer stem-like cell subpopulations. We identified SNORD44 as a suitable housekeeping gene for qPCR analysis comparing normal and cancer cells. However, this small nucleolar RNA was not a useful normalizer for cancer stem-like cell subpopulations versus subpopulations without stemness properties. In addition, we show for the first time that hsa-miR-24c-3p is the most stable normalizer for comparing these two subpopulations. Also, we have identified by bioinformatic and qPCR analysis, different miR expression patterns in colon cancer versus non tumour cells using the previously selected suitable normalizers. Our results emphasize the importance of select suitable normalizers to ensure the robustness and reliability of qPCR data for analyzing miR expression.