Real Time PCR analysis was carried out as described [12 (link)]. Primers for CCNB1, PCNA and DEPP were those utilized by Wang et al. [26 (link)], Primers for DDIT4 were designed by Primer 3 (http://primer3.ut.ee/ ) and their sequence is: FW-GGTCACTGAGCAGCTCGAA; REV-CCTGGACAGCAGCAACAGT. Relative quantification of each target gene transcript was obtained using comparative Ct method. Reference genes (ATP5b, SDHA1, and CYC1) were selected using GeNorm (PrimerDesign Ltd, Southampton, UK). For each cDNA, the duplicate Ct values were averaged and normalized (geometric mean). The copy number was expressed relative to a calibrator sample using the 2−(ΔΔCt ± SD) method.
CHIP was performed utilizing the Epitect® ChIP One Day Kit (Qiagen, Milano, Italy) utilizing the technical conditions reccomended by the manufacturer. Immunoprecipitation was performed with an anti H3K4me3 Polyclonal Antibody (Epigentek, Farmingdale NY, USA). As irrelevant antibody we utilized a chicken alpha-GFP Antibody (Invitrogen – Thermo Fisher, Waltham, MA, USA). Evaluation of the immunoprecipitate was performed by qPCR utilizing the primers described in Ref. [26 (link)]
CHIP was performed utilizing the Epitect® ChIP One Day Kit (Qiagen, Milano, Italy) utilizing the technical conditions reccomended by the manufacturer. Immunoprecipitation was performed with an anti H3K4me3 Polyclonal Antibody (Epigentek, Farmingdale NY, USA). As irrelevant antibody we utilized a chicken alpha-GFP Antibody (Invitrogen – Thermo Fisher, Waltham, MA, USA). Evaluation of the immunoprecipitate was performed by qPCR utilizing the primers described in Ref. [26 (link)]
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