Quantitative Analysis of miRNA Expression

Total RNA was extracted using the Zymo Quick-RNA MiniPrep Kit according to the manufacturer’s protocol. For pri- and pre- miRNAs and mRNAs, RT was performed using 200 ng of RNA with a Qscript cDNA Synthesis Kit (QuantaBio). For mature miRNAs, the RT reaction was performed using 200 ng of RNA with the miScript II RT Kit in a total volume of 20 μl (Qiagen). Subsequent qPCR analysis (see Supplementary Table 2 for primer sequences and Supplementary Figs. 2–7 for primer validation) using Power SYBR Green Master Mix (Life Technologies) or a TaqMan assay and a Applied Biosystems QS5 384-well PCR system (software v.1.3.0). For mature miR-155 levels, TaqMan assays were performed using the ipu-miR-155 Taqman Assay (Thermo Fisher Scientific, 467534). Data were analysed using the ΔΔC_t method as described previously^{13 (link)}.