Abstract
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited renal disorder, characterized by renal cyst development leading to end-stage renal disease. Although the appropriate choice of suitable reference is critical for quantitative RNA analysis, no comparison of frequently used “housekeeping” genes is available. Here, we determined the validity of 7 candidate housekeeping genes (Actb, Actg1, B2m, Gapdh, Hprt, Pgam1 and Ppia) in kidney tissues from mouse models orthologous to ADPKD, including a cystic mice (CY) 10–12 weeks old (Pkd1flox/flox:Nestincre/Pkd1flox/−:Nestincre, n = 10) and non-cystic (NC) controls (Pkd1flox/flox/Pkd1flox/-, n = 10), Pkd1-haploinsufficient (HT) mice (Pkd1+/−, n = 6) and wild-type (WT) controls (Pkd1+/+, n = 6) and a severely cystic (SC) mice 15 days old (Pkd1V/V, n = 7) and their controls (CO, n = 5). Gene expression data were analyzed using six distinct statistical softwares. The estimation of the ideal number of genes suggested the use of Ppia alone as sufficient, although not ideal, to analyze groups altogether. Actb, Hprt and Ppia expression profiles were correlated in all samples. Ppia was identified as the most stable housekeeping gene, while Gapdh was the least stable for all kidney samples. Stat3 expression level was consistent with upregulation in SC compared to CO when normalized by Ppia expression. In conclusion, present findings identified Ppia as the best housekeeping gene for CY + NC and SC + CO groups, while Hprt was the best for the HT + WT group.
Highlights
Autosomal dominant polycystic kidney disease (ADPKD) is manifested by bilateral development of multiple fluid-filled epithelial-derived cysts[1,2]
To elucidate this issue and bring appropriate information to PKD experimental studies involving kidney transcriptional profiles, we aimed to identify the most stable housekeeping controls from a panel, namely seven candidate genes commonly used as endogenous controls in kidney disease, not limited to Autosomal Dominant Polycystic Kidney Disease (ADPKD): actin beta (Actb), Actg[1], B2m, Glyceraldehyde-3-phosphate dehydrogenase (Gapdh), Hprt, Pgam[1], and Ppia., to be considered in kidney Reverse transcription quantitative polymerase chain reaction (RT-qPCR) obtained from Pkd1-deficient mouse models
Raw RT-qPCR data in renal tissue were obtained for the Actb, Actg[1], B2m, Gapdh, Hprt, Pgam[1] and Ppia candidate housekeeping genes for each of the Pkd1-deficient mouse models
Summary
Autosomal dominant polycystic kidney disease (ADPKD) is manifested by bilateral development of multiple fluid-filled epithelial-derived cysts[1,2]. It is the most common Mendelian disorder of the kidneys, affecting 3–5:10,000 people w orldwide[3] and the leading monogenic cause of end-stage kidney d isease[4]. To provide accurate expression analysis this method requires expression normalization of the genes of interest to a reference gene that is stable and not affected by experimental conditions In this scenario, expression stability is a major criterion for housekeeping gene selection[14]. Determining the stability of genes known to be involved in ADPKD and used as housekeeping is important
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