Identification of stably expressed reference small non-coding RNAs for microRNA quantification in high-grade serous ovarian carcinoma tissues

Abstract

MicroRNAs (miRNAs) belong to a family of small non-coding RNAs (sncRNAs) playing important roles in human carcinogenesis. Multiple investigations reported miRNAs aberrantly expressed in several cancers, including high-grade serous ovarian carcinoma (HGS-OvCa). Quantitative PCR is widely used in studies investigating miRNA expression and the identification of reliable endogenous controls is crucial for proper data normalization. In this study, we aimed to experimentally identify the most stable reference sncRNAs for normalization of miRNA qPCR expression data in HGS-OvCa. Eleven putative reference sncRNAs for normalization (U6, SNORD48, miR-92a-3p, let-7a-5p, SNORD61, SNORD72, SNORD68, miR-103a-3p, miR-423-3p, miR-191-5p, miR-16-5p) were analysed on a total of 75 HGS-OvCa and 30 normal tissues, using a highly specific qPCR. Both the normal tissues considered to initiate HGS-OvCa malignant transformation, namely ovary and fallopian tube epithelia, were included in our study. Stability of candidate endogenous controls was evaluated using an equivalence test and validated by geNorm and NormFinder algorithms. Combining results from the three different statistical approaches, SNORD48 emerged as stably and equivalently expressed between malignant and normal tissues. Among malignant samples, considering groups based on residual tumour, miR-191-5p was identified as the most equivalent sncRNA. On the basis of our results, we support the use of SNORD48 as best reference sncRNA for relative quantification in miRNA expression studies between HGS-OvCa and normal controls, including the first time both the normal tissues supposed to be HGS-OvCa progenitors. In addition, we recommend miR-191-5p as best reference sncRNA in miRNA expression studies with prognostic intent on HGS-OvCa tissues.

Keywords: qPCR; HGS-OvCa; endogenous reference; normalization; sncRNA.

Figure 1

Expression levels of candidate reference sncRNAs in HGS‐OvCa (hatched boxes) and normal (open boxes) tissues. Values are given as calibrated normalized relative quantities (CNRQ). Boxes indicate IQR for the data of unmatched samples in each group. Error bars represent range of values.

Figure 2

geNorm output charts. (A) Average expression stability value (M) of reference sncRNAs calculated at each step during stepwise exclusion of the least stable expressed reference. Starting from the least stable at the left, the sncRNA are ranked according to increasing expression stability, ending with the two most stable sncRNA on the right. (B) Normalization factor based on the pairwise variation (V), representing the levels of variation in average reference sncRNA stability with the sequential addition of each reference to the equation.