Specific mircury lna mirna pcr assays

To confirm miRNA-seq results, we used quantitative real-time PCR (qPCR) to measure the expression of selected miRNAs in the mouse kidney. Reverse transcription reaction (cDNA synthesis) was performed using the miRCURY LNA RT kit (Qiagen), and real-time PCR with specific miRCURY LNA miRNA PCR Assays (Qiagen) was carried out with a QuantStudio 7 Real-Time PCR detection system (Applied Biosystems) using miRCURY LNA SYBR Green PCR kit (Qiagen). Total reaction volume for qPCR reaction was 10 μL. Forty cycles at 95 °C for 10 s and 56 °C for 60 s min were performed, followed by melting curve analysis. Relative miRNA levels were calculated by standard formulae (ΔΔCt method) using miR-30a-3p and miR-30b-5p as endogenous controls. Only candidates with high expression levels were subjected to stability analysis (expressed in 100% of samples and expressed in the first quartile (Q1)). Endogenous controls were selected combining statistical tools like NormFinder, geNorm, Best-Keeper and Delta Cq/Ct algorithms that are integrated within the RefFinder software [88 (link)]. specific miRCURY LNA miRNA PCR Assays (Qiagen) were as follows: miR-5099 (YP02107868), miR-223-3p (YP00205986), miR-551b-3p (YP00204067), miR-21a-3p (YP00205400), miR-146a-3p (YP02115408), miR-30a-3p (YP00204457) and miR-30b-5p (YP00204765).