Mircury lna microrna system control primer set

RNA was extracted from cultured cells using TRIzol reagent (Ambion) followed by isopropanol-alcohol precipitation (RNeasy Mini Kit, Qiagen) before quantitation. RNA was then converted to cDNA with High Capacity cDNA Reverse Transcription kit (Applied Biosystem) according to the manufacturer’s protocol. Gene expression analysis was carried out using GAPDH or β-Tubulin reference gene. Primers for mouse CyclinD1 (CycD1), Myogenin (Myog), mouse myogenic differentiation 1 (MyoD1), Pax7, Follistatin (FST), Myosin Heavy Chain 2 (Myh2), β-Catenin, Calcineurin, Utrophin, Transforming growth factor beta (TGF-β), Cullin-1, MRF4, MuRF-2, FBXW7 and β-TrCP are reported in Supplementary Table S1. Transcript levels were assessed using the Bio-Rad CFX machine according to the manufacturer’s instructions and each experiment was repeated three times using independent RNA samples. For microRNAs quantitative reverse transcription–polymerase chain reaction, primers for mature miR-206, miR-133, miR-27b, the internal control U6, and miR-16 were used according to the manufacturer’s protocol (MiRCURY LNA microRNA system control primer set, Exiqon, Vedbaek, Denmark).

The data from each cDNA sample were normalised for real-time qPCR experiments using the mouse housekeeping gene Gapdh (glyceraldehyde 3-phosphate dehydrogenase). The specific primers used for amplification of Gapdh (NM_001289726.2), Cripto-UTR (NM_011562.2), Cripto (NM_011562.2), Nkx 2.5 (NM_008700.2), Gata-4 (NM_001310610.1), Apj (NM_011784.3), and Mlc-2 (NM_010861.4) were designed based on the nucleotide sequences downloaded from the NCBI database using Primer3WEB v.4.0.0 (see Table 2). For microRNA quantitative reverse transcription–polymerase chain reaction, primers for mature miR-1 and the internal control U6 were used according to the manufacturer’s protocol (MiRCURY LNA microRNA system control primer set, Exiqon) [69 (link)].
Calculations of relative expression levels were performed using the 2^−ΔΔCt method [70 (link),71 (link)]. All analysis was performed in triplicate to guarantee the accuracy of results.