Lightcycler 480 2.0 real time pcr system

Total RNA from MEFs was isolated using TRIzol Reagent (Life Technologies) according to the manufacturer’s instructions. Contaminant genomic DNA was eliminated with TURBO DNA-free kit (Ambion). Reverse transcription was carried out using 1 μg DNA-free RNA and 50 μM random hexamers, 20 U of RNase Out and 100 U of RevertAid reverse transcriptase (Life Technologies). Complementary DNA reactions were used as templates for PCR reactions. Real-time PCR was performed using the light cycler-DNA MasterPLUS SYBR Green I mix (Thermo Scientific) supplemented with 0.5 μM of specific primer pairs (listed in Supplementary Table 2). Real-time qPCRs were run on a light cycler rapid thermal system (Light Cycler 480 2.0 Real time PCR system, Roche) with 20 s of denaturation at 95 °C, 20 s of annealing at 60 °C and 20 s of extension at 72 °C for all primers, and analysed by the comparative CT (▵C_t) method.

Genomic DNA (200 ng) was digested at 37°C for 4 h with 10 U of the methylation-sensitive enzyme AciI, or NcoI (New England Biolabs) which does not have cutting sites in our regions of interest and served to normalize the data. The endonucleases were subsequently inactivated by incubation at 65°C for 20 min. Real-time PCR was carried out using the light cycler-DNA MasterPLUS SYBR Green I mix (Roche) supplemented with 0.5 μM specific primer pairs and with 2 μL of digested DNA. Real-time quantification PCR were run on a light cycler rapid thermal system (LightCycler®480 2.0 Real time PCR system, Roche) with 20 sec of denaturation at 95°C, 20 sec of annealing at 65°C and 20 sec of extension at 72°C for all primers, and analyzed by the comparative CT (∆CT) method according to the formula: methylation (%) = E^(∆CT) × 100 where E represents PCR efficiency and ∆CT = CT_sample (AciI digest) - CT_sample (NcoI digest). Sequences of primers within CpG islands at germline gene promoters are shown in Additional file
1. Each data shown on histograms is the mean result of qPCR analysis on at least three independent experiments performed on at least three independent genomic extractions.