Validation of Methylated Genes and miRNA Expression

For the validation of methylated genes expression, we used primers for Socs1, Ep300, p16, Mgmt and Dapk (QuantiTect Primer Assay, Qiagen); for the evaluation of miR-466e-5p gene targets expression, we used primers for Zfp704, Cplx2 and Cnot7[41] (link), [42] (link); for the validation of miRNA microarrays, we used primers for miR-466e-5p, miR-21-3p and miR-185-3p (MystiCq microRNA qPCR Assay Primer, Sigma-Aldrich). Mouse liver RNA was reverse-transcribed using the RT2 first strand kit (SABiosciences, Frederick, MD, USA) for gene expression experiments and MystiCq microRNA cDNA Synthesis Mix (Sigma-Aldrich) for miRNA expression experiments. Then quantitative real-time PCR reactions were performed in triplicate with an Applied Biosystems 7300 Real-Time PCR system (Life Technologies), using the RT2 SYBR Green PCR Master Mix (SABiosciences) for gene expression experiments and the MystiCq microRNA SYBR Green qPCR ReadyMix for miRNA expression experiments, according to the manufacturers’ instructions.