Identification of housekeeping genes for microRNA expression analysis in kidney tissues of Pkd1 deficient mouse models

Polycystic kidney disease is a complex clinical entity which comprises a group of genetic diseases that leads to renal cyst development. We evaluated the most suitable housekeeping genes for microRNA expression by RT-qPCR analyses of kidney tissues in Pkd1 -deficient mouse models from a panel of five candidates genes ( miR-20a , miR- 25, miR-26a , miR-191 and U6 ) and 3 target genes ( miR-17 , miR-21 and let-7a ) using samples from kidneys of cystic mice ( Pkd1 flox/flox : Nestin cre , CY), non-cystic controls ( Pkd1 flox/flox , NC), Pkd1 -haploinsufficient ( Pkd1 +/− , HT), wild-type controls ( Pkd1 +/+ , WT), severely cystic mice ( Pkd1 V/V , SC), wild-type controls (CO). The stability of the candidate genes was investigated using NormFinder, GeNorm, BestKeeper, DataAssist, and RefFinder software packages and the comparative ΔCt method. The analyses identified miR-26a as the most stable housekeeping gene for all kidney samples, miR-20a for CY and NC, miR-20a and miR-26a for HT and WT, and miR- 25 and miR-26a for SC and CO. Expression of miR-21 was upregulated in SC compared to CO and trends of miR-21 upregulation and let-7a downregulation in CY and HT compared to its control kidneys, when normalized by different combinations of miR-20a , miR- 25 and miR-26a . Our findings established miR-20a , miR- 25, and miR-26a as the best housekeeping genes for miRNA expression analyses by RT-qPCR in kidney tissues of Pkd1 -deficient mouse models.