Mastercycler realplex apparatus

Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis was described elsewhere.^{35 (link)} Briefly, the cells were treated with different concentrations of TQ for 24 h. Then, total RNA was purified and subjected to reverse transcription using Oligo(dt) (Sigma, Steinheim, Germany) and Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitative real-time PCR was performed using the LightCycler 480 SYBR Green I Master Kit (Roche Diagnostics, Indianapolis, Indiana, USA) and the Mastercycler Realplex apparatus (Eppendorf, Montesson, France). The results were normalized with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The sequences of the primers for PCR amplification were UHRF1 (sense: 5′-GTCGAATCATCTTCGTGGAC-3′, antisense: 5′-AGTACCACCTCGCTGGCAT-3′), DNMT1 (sense: 5′-GCACAAACTGACCTGCTTCA-3′, antisense: 5′-GGCCTTTTCACCTCCATCAA-3′), HDAC1 (sense: 5′-GACAAGGCCACCCAATGAAG-3′, antisense: 5′-GCTTGCTGTACTCCGACATG-3′), G9a (sense: 5v-GGAGAAGTGACCCTGACGAA-3′, antisense: 5′-CCTCTTCCTCCTCCTCCTCT-3′), and GAPDH (sense: 5′-GGTGAAGGTCGGA-GTCAAC-3′, antisense: 5′AGAGTTAAAAGC-AGCCCTGGTG-3′). Amplicons were size controlled on agarose gel, and purity was assessed by analysis of the melting curves at the end of the RT-PCR reaction.

Total RNAs were purified from subconfluent MEFs using standard methods and subjected to reverse transcription using random primers (Promega) and the SuperScript II reverse transcriptase (Invitrogen). Real-time quantitative PCR was done with the LightCycler 480 SYBR Green I Master kit (Roche) and the Mastercycler Realplex apparatus (Eppendorf). PCRs were performed with the oligonucleotide pairs 5′-GCCAGATGTGCTCAGTTTCC-3′ and 5′-CTGCCTCATAGCCTGGATCA-3′ for Tdg and 5′-GGCTGTATTCCCCTCCATCG-3′ and 5′-CCAGTTGGTAACAATGCCATGT-3′ for Actb. Results were normalized to Actb.