RNA isolation and qPCR.

Total RNA and miRNA-enriched fractions were isolated from aortic tissue using RNeasy and miRNeasy kits, respectively (Qiagen, Valencia, CA), per manufacturer’s instructions. To avoid genomic DNA contamination, DNase digestion was performed using RNase free DNase kit (Qiagen, Valencia, CA), per manufacturer’s instruction.

To characterize the integrity of the isolated RNA, spectrophotometric evaluation was performed using Nanodrop (Thermo Scientific, Wilmington, DE). Only RNA samples with an A260 (absorbance at 260 nm) value greater than 1.80 were used for further experiments. The ratio of the readings at 260 nm and 280 nm (A260/A280) was also measured to check the purity of the isolated RNA. For further and more accurate purity and integrity estimation of the isolated RNA, samples were profiled by the Bioanalyzer 2100 (Agilent Technologies). Only high-quality RNA, A260/A280 ~2.00 and RIN >8, was used for these experiments.

For samples that passed these tests of purity and integrity, the RNA was reverse transcribed using miScript Reverse Transcription Kit (Qiagen, CA). For mRNA we used HiFlex and for miRNA we used HiSpec buffers, respectively. PCR was performed using a miScript SYBR green PCR kit (Qiagen, Valencia, CA) and detected with a CFX96 detector (Bio-Rad). All reactions were performed in triplicate. Quantitative PCR (qPCR) was performed with primers for miR-181a, miR-181b, let-7a, miR-126-3p, and pre-miR-181b (Qiagen, CA). Additionally, qPCR was performed for Translin (TN) and Trax (TX) from the mRNA fraction of cDNAs. The primer sequences used for qPCR analysis of TN and TX mRNAs are shown in Table 1.

Primer sequences used for qPCR analysis of TN and TX mRNAs

qPCR, quantitative PCR; TN, translin; TX, trax.