Connect real time pcr

For siRNA transfection, MCF7 cells in 6-well tissue culture plates were transiently transfected with the HiPerfect transfection reagent (Qiagen) using 10 nM a scrambled siRNA (AllStar, CtS), a siRNA specific for CXXC5 (siRNA#10; FlexiTube GeneSolution, Qiagen), as we described previously^{19 (link),21 (link)}, and/or a siRNA pool specific to MeCP2 (sc-35892, SCBT). To equalize the total amount of siRNA (20 nM) used in co-transfection experiments, 10 nM gene-specific siRNA was used together with 10 nM CtS. Isolated total RNA was used for the cDNA synthesis (The RevertAid First Strand cDNA Synthesis Kit, ThermoFisher). The SYBR Green Mastermix (BioRad, Hercules, CA, USA) and gene-specific primers (Supplementary Information, Table S2) were used for qPCR reactions on BioRad Connect Real-Time PCR.
For the normalization of results, we used the expression of RPLP0 (60S acidic ribosomal protein P0), as we described previously^{95 (link)}. The relative quantification of gene expressions was assessed with the comparative 2^-ΔΔCT method^{96 (link)}. For qPCR experiments, MIQE Guidelines were followed^{97 (link)}.

Isolated total RNA from cells was used for the cDNA synthesis (The RevertAid First Strand cDNA Synthesis Kit, Thermo-Fisher). The SYBR Green Mastermix (BioRad, Hercules, CA, USA) and gene-specific primers (Supplementary Table S1) were used for RT-qPCR reactions on BioRad Connect Real-Time PCR, as we described previously^{4 (link)}. For the normalization of results, we used the expression of RPLP0 (60 S acidic ribosomal protein P0), a reference gene for the normalization of E2-regulated genes in breast carcinomas^{66 (link)}. The relative quantitation of gene expressions was assessed with the comparative 2^-ΔΔC_T method^{67 (link)}. During the RT-qPCR experiments, MIQE Guidelines were followed^{68 (link)}.