Mmlv reverse transcriptase and random hexamers

Biopsies were homogenised using a 5 mm stainless steel ball-bearing in a Retsch^® Bead Mill MM 301 for 4 min, at 30 oscillations per second. Total RNA from biopsy homogenate and lysed fibroblasts was isolated using Nucleospin^® RNA isolation kits (Machery-Nagel, UK) following the manufacturer's instructions. RNA concentration was measured using a NanoDrop™ (ND-1000, ThermoScientific, UK). RNA was diluted in water to 100 ng/μl, and cDNA synthesised using M-MLV Reverse Transcriptase and random hexamers (Promega, Madison, USA) according to manufacturer's instructions. To identify any residual genomic DNA contamination, samples with and without reverse transcription were PCR amplified for GAPDH, an abundant transcript, using forward (5′-CCACCAACTGCTTGGCCCCC-3′) and reverse (5′-GGACACGTTGGGGGTGGGGA-3′) primers. This was performed using DreamTaq Polymerase (Fermentas Life Sciences, York, UK), 10 μM of each primer, 10 μM dNTP mix (Promega, Madison, USA) and 25 ng of cDNA. The PCR program consisted of a 95 °C denaturation for 3 min, 25 cycles at 95 °C for 10 s, 55 °C for 1 min, 72 °C for 1 min, and a final extension at 72 °C for 10 min.