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

Abstract

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 (Pkd1flox/flox:Nestincre, CY), non-cystic controls (Pkd1flox/flox, NC), Pkd1-haploinsufficient (Pkd1+/−, HT), wild-type controls (Pkd1+/+, WT), severely cystic mice (Pkd1V/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.

Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models

“…The results yielded by the two analyses did not significantly differ, showing similarity and stability of genetic background between both model pairs. Of note, this pattern was also observed between such model pairs in a previous study of ours directed to identifying housekeeping genes for microRNA expression analysis 21 . In this context, in the current study we analyzed this cystic model merging Pkd1 flox/flox : Nestin cre and Pkd1 flox/− : Nestin cre mice in the CY group and Pkd1 flox/flox and Pkd1 flox / - in the NC group.…”

Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidney tissues of Pkd1 deficient mouse models

“…Of note, this pattern was also observed between such model pairs in a previous study of ours directed to identifying housekeeping genes for microRNA expression analysis. 21 In this context, in the current study we analyzed this cystic model merging Pkd1 ox/ ox :Nestin cre and Pkd1 ox/-:Nestin cre mice in the CY group and Pkd1 ox/ ox and Pkd1 ox /in the NC group.…”

“…The mice were genotyped using speci c PCR reactions. 21 The CY mice, homozygous for a Pkd1oxed allele (Pkd1 ox ) or compound heterozygous Pkd1 ox/-, display a mosaic pattern of full gene inactivation induced by a Nestin-Cre transgene through excision of exons 2-4 (Pkd1 ox/ ox :Nestin cre ). 38,58,59 60 The HT model is heterozygous for a Pkd1 null allele, characterized by early transcriptional interruption, 61 60 and develops no renal cysts by 12 weeks of age.…”

“…Both kidneys were collected and stored at − 80°C for further use. 21 Housekeeping genes Using the terms "genes" and "polycystic and reverse transcriptase polymerase chain reaction" in the PubMed search, we found seven articles employing tissue samples from human ADPKD and animal ADPKD-orthologous kidneys. We also included housekeeping genes selected from other tissues in the literature, 24,31,34−37 38 namely Actb, Actg1, B2m, Gapdh, Hprt, Pgam1 and Ppia.…”

“…Total RNA was stored at -80°C until further use. 21 cDNA preparation and RT-qPCR Complementary DNA (cDNA) was synthesized from 2 µg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), according to the manufacturer`s instructions.…”