RNA Extraction and RT-qPCR Analysis

RNA was extracted using the RNeasy kit (Qiagen, Crawley, UK), according to the manufacturer’s instructions. For cDNA synthesis, 300–500 ng RNA was used in combination with 0.5 μg random primers (Roche, West Sussex, UK), 40 U RNaseOUT 0.5 μM dNTPs (Invitrogen, Paisley, UK) 1× RT Buffer (Fermentas, Cambridge, UK) and RevertAid reverse transcriptase (Fermentas, Cambridge, UK) made up to a final volume of 20 μl using RNase-free water (Qiagen, Crawley, UK). Reactions were carried out in a thermocycler with conditions—25 °C for 10 min, 42 °C for 60 min and 70 °C for 10 min. One microlitre cDNA per well on a 96-well plate (Roche) was used for RT-qPCR with SYBR Green reagent and remaining cDNA stored at −80 °C. RT-qPCRs were performed using a LightCycler 480 Instrument II (Roche, West Sussex, UK). Gene expression was normalised to Hprt and relative expression calculated by the ΔΔC_T method [40 (link)]. Each RT-qPCR contained 1× buffer, 0.4 mM dNTPs, 50 μM primers (Additional file 1: Table S1), 0.01 U Taq DNA polymerase (Invitrogen, Paisley, UK) and nuclease-free water (Qiagen, Crawley, UK). Four primer sets for Dnmt1, Dnmt3a, Dnmt3b [47 (link)] and Hprt [13 (link)] were used. The general thermocycler conditions are as follows—94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 63 °C for 1 min, 72 °C for 1 min with a final elongation step of 72 °C for 4 min.