Kidney Gene Expression Analysis

Snap-frozen mouse kidneys were homogenised using MagNa Lyser technology (Roche). Total RNA was isolated using TRI Reagent (Sigma-Aldrich). cDNA synthesis was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche), and qPCR was done using 2× FastStart SYBR Green Master (Roche) according to the manufacturer’s protocol. Alternatively, it was performed at GenomeScan (GenomeScan B.V.) using the 96.96 BioMark™ Dynamic Array for real-time PCR (Fluidigm Corporation), as previously described [5 ]. Gene expression was normalised to the geometric mean of three housekeeping genes (Rplp0, Hnrnpa2b1, Ywhaz) for Fluidigm data and Hprt for SYBR-Green data. The output of the Fluidigm assay was normalised and converted into Ct values (cycle threshold). For each transcription factor, a two-way ANOVA was conducted to compare the genotype (PKD vs WT) and the treatment (PBS vs DCVC) effects for each age-matched time points. The computation was made using the Limma package [10 (link)] in R. A list of primer sequences and TaqMan assays can be found in Supplementary Table 3.