Identification of Stable Housekeeping Genes for miRNA Expression Studies in a Mouse Unilateral Ureteral Obstruction Model.

Recently, several studies have aimed to establish the role of microRNAs (miRNAs) in the unilateral ureteral obstruction (UUO) model. Therefore, it is essential to identify the best housekeeping genes (HKG) to correctly estimate the expression levels of miRNAs. The present study aimed to identify suitable HKG to normalize the expression of miRNAs by RT-qPCR in kidney samples from the UUO mice model. We analyzed the stability of twelve endogenous reference genes of small non-coding RNAs (Snord61, Snord68, Snord72, Snord95, Snord96a, U6, let-7e-5p, let-7i-3p, miR-15b-5p, miR-16a-5p, miR-26a-5p, and miR-30c-5p) by using four software packages: NormFinder, GeNorm, ΔCt method, and BestKeeper. The optimal number of genes was calculated using GenEx software analysis. To validate the best HKG, we normalized the expression of miR-18a-5p, miR-21a-3p, and miR-29b-3p. In silico analysis revealed that Snord61, Snord68, and Snord72 were the most stable HKG between the groups. Using GenEX software and Pearson's correlation, we determined that the combination of Snord61 and Snord68 or the combination of Snord68 and Snord72, provided the best HKG association. These results along with the correlation analyses establish that the association of Snord68 and Snord72 is the best choice for miRNA expression analysis by RT-qPCR in the UUO model.