267 Generation of Human iPSC lines derived from PLN R14del Cardiomyopathy Patients 10 with PBS for 5 minutes and nuclei were stained using 1 µg/ml Hoechst (Life Technologies) for 15 minutes. Coverslips were mounted using Fluoromount-G (Southern Biotech) and images were acquired using Leica DMi8 confocal microscope. RNA isolation and cDNA synthesis. Total RNA was isolated from cells using TriZol (Ambion) according to the manufacturer’s instructions. DNase treatment was carried out using RNaseFree DNase Set (Qiagen). Two units of DNase were used to treat 1 µg of RNA for 15 minutes at 42°C and DNase was inactivated for 5 minutes at 65°C. One µg of DNase-treated RNA was converted to cDNA using the iScript™ cDNA Synthesis Kit (Quatetect). After reverse transcription (15 min 42°C) and RT inactivation (3 min 95°C), the cDNA was diluted 20X with MilliQ before qRT-PCR. qRT-PCR. Relative gene expression was determined by qRT-PCR using the (5′–3′) primers listed in Table 2. The following cycling parameters were used for qPCR: Incubation at 95°C for 3 minutes, followed by 40 three-step cycles, each consisting of 95°C for 10 seconds (denaturation), 58°C for 10 s (annealing), and 72°C for 30 s (elongation), followed by melting curve analysis. qPCR was performed using SYBR Green Master Mix (Biorad) in a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad). All experiments were performed in duplicate and values were normalized to the housekeeping gene RPL32. The ΔΔCt was determined by comparing it to control gene expression and the relative fold increase in expression was calculated by 2^-ΔΔCt.
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