Cardiac Loops qRT-PCR Analysis

Cardiac loops isolated from experimental and control (CFDA) embryos (Supplementary Figure S2) were subjected to qRT-PCR analysis following MIQE guidelines [72 (link),73 (link),74 (link)]. RNA was extracted and purified using the ReliaPrep RNA Cell Miniprep System Kit (Promega) according to the manufacturer’s instructions. For mRNA expression measurements, 1 μg of total RNA was used for retro-transcription with a Maxima First Strand cDNA Synthesis Kit for qRT-PCR (Thermo Scientific). Real-time PCR experiments were performed with 2 μL cDNA, Go Taq qPCR Master Mix (Promega) and corresponding primer sets (Supplementary Table S1). For microRNA expression analyses, 20 ng of total RNA was used for retro-transcription with Universal cDNA Synthesis Kit II (Exiqon) and the resulting cDNA was diluted 1/80. Real-time PCR experiments were performed with 1 μL of diluted cDNA, Go Taq qPCR Master Mix (Promega) as well. All qPCRs were performed using a CFX384TM thermocycler (Bio-Rad) following the manufacture’s recommendations. The relative expression of each gene was calculated using Gusb and Gadph as internal controls for mRNA expression analyses and 5S and 6U for microRNA expression analyses, respectively [75 (link)]. Each PCR reaction was carried out in triplicate and repeated in at least three distinct biological samples to obtain representative means.