Sybr green based gene expression analyses

Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151). For qPCR analysis of miR-151-5p and miR-133a, 100 nanograms of Trizol extracted RNA was reverse-transcribed using miRNA Reverse Transcription kit (Life technologies) followed by qPCR using miRNA Taqman probes (002642 from Life Technologies). To look for the endogenous E2f6 levels on overexpression of miR-151-5p by qPCR, MEF cells were co-transfected with GFP and Sh-151-5p and were subsequently FACS sorted for GFP. For the ChIRP experiment, the pulled down RNAs were reverse-transcribed using Thermoscript (Life technologies) and subjected to SYBR-Green based gene expression analyses (Qiagen) using primers specific for pre-miR-151, pre-miR-124 and U6 (see Supplementary Table 4 for sequences) as described previously^{45 (link)}. Quantitative RT-PCR was performed on a CFX384 Real-Time system (BioRad). Fold-change was detected using delta-delta-cT calculations and normalized to mouse beta actin and U6 in gene expression and miRNA expression analyses respectively.