Faststart sybr green master

Methylation levels for the C9orf72 promoter in patients were determined as previously described (45 (link)). Briefly, genomic DNA from the cerebellum was extracted using the Qiagen DNeasy Blood and Tissue kit and subject to overnight digestion with HhaI and HaeIII (double-digested) or just HaeIII (mock) alone. A small aliquot of DNA was amplified using primers flanking the HhaI cutsite within the C9orf72 promoter region using 2x FastStart SYBR Green Master (Roche) on the ABI StepOnePlus machine. The difference in cycles to threshold amplification between double and mock digested DNA was calculated as methylation values. Spearman’s correlation was calculated using gene counts for each gene and methylation values for each C9orf72 mutation case. R package ‘lsr’ with ‘correlate’ function (with options corr.method =“spearman” and p.adjust.method=“fdr”) using the Spearman’s correlation and FDR adjusted p-values was used. Only correlations from genes with HUGO gene symbols were calculated and plotted against gene fold change for each gene that was calculated by DESeq2. This was done using both significantly differentially expressed genes linked to the C9orf72 mutation (DESeq2 FDR p-value < 0.05) and genome-wide using all expressed genes.

Gene expression analysis by quantitative PCR (qPCR) was performed as described previously (Happe et al., 2011). Briefly, cDNA synthesis of total RNA was done using Transcriptor First Strand cDNA Synthesis Kit (Roche, Almere, The Netherlands; #04897030001) according to the manufacturer's protocol. Quantitative PCR was done in triplicate on the LightCycler 480 II (Roche) using 2x FastStart SYBR‐Green Master (Roche; #04913914001) according to the manufacturer's protocol. Data was analyzed with LightCycler 480 Software, Version 1.5 (Roche). Gene expression was calculated using the 2^−ΔΔCt method (Livak & Schmittgen, 2001) and normalized to the housekeeping gene Hprt, giving the relative gene expression. For primer sequences see Supplementary Table S1. Mean gene expression and standard deviation (SD) of the different treatment groups were calculated. Differences between fluid shear stress treated cells and static controls were tested using one sample t‐tests. One‐way analysis of variance (ANOVA) was used when cells were exposed for a different time or to a different flow rate. Two‐way analysis of variance (ANOVA) was used, when the shear stress response was compared to a second treatment. The ANOVA was followed by post‐hoc Fisher's LSD multiple comparison, if the overall ANOVA F‐test was significant. p < 0.05 was considered to be statistically significant.

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.

Total RNA was isolated from cultured cells using TRI Reagent (Sigma–Aldrich; #T9424) according to manufacturer’s protocol. Gene expression analysis was performed by quantitative PCR (qPCR) as described previously [38 (link)]. Briefly, cDNA synthesis was done using Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science; #04897030001) according to the manufacturer’s protocol. Quantitative PCR was done in triplicate on the LightCycler 480 II (Roche) using 2× FastStart SYBR-Green Master (Roche; #04913914001) according to the manufacturer’s protocol. Data was analyzed with LightCycler 480 Software, Version 1.5 (Roche). Gene expression was calculated using the 2^− ΔΔCt method as described previously [39 (link)] and normalized to the housekeeping gene Hprt, giving the relative gene expression. Mean gene expression and standard deviation of the different treatment groups were calculated. For primer sequences see Supplementary Material 1, Table S1.

Snap‐frozen kidneys were homogenised using Magnalyser technology (Roche, Basel, Switzerland). Total RNA was isolated using Tri‐Reagent (Sigma‐Aldrich). cDNA synthesis was performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche), and qPCR performed using ×2 FastStart SYBR‐Green Master (Roche) according to the manufacturer's protocol. Primer sequences are provided in supplementary material, Table S1. Levels of mRNA were normalised to Hprt and fold‐change was used for representation in the graphs.