Phase lock gel heavy tubes

Total RNA was obtained using the Trizol reagent (Thermo Fisher Scientific). Briefly, samples were incubated for 5 min at RT, 200 μL of chloroform was added, mixed, and incubated for 3 min at RT. Samples were centrifuged in Phase Lock Gel Heavy tubes (VWR, Radnor, PA, USA) at 12,000× g at 4 °C for 15 min. Afterwards, the aqueous phase was collected to precipitate RNA by adding 450 μL of isopropanol 100%. After centrifugation at 12,000× g at 4 °C for 10 min, the pellet was washed with ethanol 75%, resuspended in RNase free water and kept at −80 °C until further use.

Total RNA was extracted using TRIzol reagent (Ambion, Warrington, UK). Cells in 12‐well plates were lysed in 600ml TRIzol reagent and transferred into Phase Lock Gel Heavy tubes (5 prime, VWR, Leicestershire, UK) according to the manufacturer's instructions. RNA purity and quantity were assessed by Nano Drop 1000 Mass spectrometer (Fisher Scientific). Ratios of A260/A280 between 1.8 and 2 were considered to be of high purity and used for cDNA synthesis. Possible contaminating DNA was removed and cDNA prepared from 1 μg RNA using QuantiTect Reverse Transcription Kit (Qiagen, West Sussex, UK) according to the manufacturer's instructions. qRT‐PCR was performed with a Rotor‐Gene 6000 thermal cycler (Qiagen) using Brilliant III Ultra‐Fast SYBR Green qPCR Master mix (Stratagene, Agilent Technologies, Cheshire, UK) and primer pairs as listed in the Table 1. PCR conditions consisted of 1 cycle of 95°C for 3 min. and 40 cycles of 95°C for 10 s. and 60°C for 10 s. followed by melting analysis of 1 cycle with gradual increase from 65 to 95°C. RPL13a was used as the housekeeping gene.