Rq1 dnase kit

RNA was isolated with TRIzol reagent (Invitrogen) followed by use of a RQ1 DNase kit (Promega) to remove potential genomic DNA contamination. Standard reverse transcription was carried out to generate cDNA.
Real-time PCR was performed on a Bio-Rad CFX touch 1000 qPCR system, and qPCR was carried out by using Bio-Rad CFX Manager software. The validated primers used are listed in Table S2. The housekeeping gene RPL13A (Curtis et al. 2010 (link)) and other validated primers were from Qiagen. The qPCR results were pooled for a gene study using Bio-Rad CFX Manager software.

For gene expression analysis, mRNA was isolated from kidney homogenates or cultured cells using TriReagent isolation reagent (#11667165001; Roche Diagnostics) according to the manufacturers recommendations. All mRNA samples were quantified by spectrophotometry and stored at −80 °C until further analysis. 1 μg of mRNA was treated with DNAse using the RQ1 DNAse kit (M6101, Promega, Madison, WI, USA) and subsequently converted to cDNA using M-MLV reverse transcriptase (M1705, Promega, Madison, WI, USA) and random hexamer primers (#SO142, Fisher scientific, Landsmeer, the Netherlands) according to the manufacturers recommendations. qPCR and subsequent analysis were performed using a Roche lightcycler with SYBR green PCR master mix (#04707516001; Roche, Almere, the Netherlands) on a Lightcycler 480 machine and corresponding software (Software release 1.5.0 (1.5.0.39), Roche, Almere, the Netherlands). Expression levels were normalized using the average expression levels of GAPDH and TBP. The following primer sequences were used: mPAR-1 forward: 5′-GTTGATCGTTTCCACGGTCT-3′reverse: 5′-ACGCAGAGGAGGTAAGCAAA-3′; mTBP forward: 5′-GGAGAATCATGGACCAGAACA-3′ reverse: 5′-GATGGGAATTCCAGGAGTCA-3′; hPAR-1 forward: 5′-GCAGGCCAGAATCAAAAGCAACAAATGC-3′ reverse: 5′-TCCTCATCCTCCCAAAATGGTTCA-3′; hTBP forward: 5′-ATCCCAAGCGGTTTGCTGC-3′ reverse: 5′-ACTGTTCTTCACTCTTGGCTC-3′.

Culture volume equivalent to OD₆₀₀ = 2 was collected, 1 ml of TRI reagent (Sigma Aldrich, St. Louis, MO) was added to the pellet and samples were stored at − 80 °C. The total RNA from the cell pellet was extracted using manufacturer’s recommendation. The RNA samples were DNase treated using RQ1 DNase kit (Promega, Madison, Wisconsin) followed by cDNA synthesis of 2 µg sample using ProtoScript^® II First Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, Massachusetts). Quantitative real time-PCR was carried out Light cycler 480 II (Roche Diagnostics, Indianapolis, IN) using LuminoCt^® SYBR^® Green qPCR ReadyMix™ (Sigma-Aldrich, St. Louis, MO) and gene specific primers. The expression of genes was normalized to the expression of 16S rRNA, which was used as the internal control. No template control reactions were set-up for each primer pair. The list of oligonucleotide primers used for RT-PCR is provided in Supplementary Table S1.