Iscript reverse transcription supermix for reverse transcription

Total RNA was isolated from 30 mg of left ventricular heart tissue using an AllPrep DNA/RNA/miRNA isolation kit (Qiagen, Germantown, MD), following the manufacturer protocol. Isolated RNA was then synthesized into cDNA using iScript Reverse Transcription Supermix for reverse transcription (Bio-Rad, Hercules, CA). Synthesized cDNA was used for real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to determine transcriptional expression of messenger RNA (mRNA) using forward and reverse primers for LOX-1, ABCA-1, ICAM-1, VCAM-1, TNF-α, IL-6, IL-1β, ET-1, MMP-9, OPG, TGF-β1, RelA, RelB, IKK-α/β, and IκB-α (Supplementary Table 1) using Bio-Rad SSo SYBR green detection (Bio-Rad), following the manufacturer’s protocol. Isolated RNA, in conjunction with the miScript II RT kit (Qiagen), produced cDNA specific to miRNA detection, following manufacturer’s instruction. Primers for detecting the transcriptional expression of miR-1, miR-21, and miR-221 using miScript SYBR Green PCR kit (Qiagen) with specific primers for each miRNA (Supplementary Table 1). Results for both mRNA and miRNA RT-qPCR were calculated/normalized, as previously described by our laboratory (Lund et al., 2009 (link), 2011). An n=7 per group were used for real time PCR analysis.