Quantitative RT-PCR Analysis of Megakaryocytic Differentiation

Total RNA was isolated using Trizol Reagent (Thermo Fisher), from Meg01 or Meg01-MEF2CKO cells treated with 40nM phorbol 12-myristate 13-acetate (PMA) or DMSO for 24 hours; CHOP10 or CHOP10-MEF2CKO HPCs after 11 days culture in megakaryocytic differentiation media,^{18 (link)} and from CB CD34⁺ derived megakaryocytes after 13 days culture in megakaryocytic differentiation media.^{19 (link)} 3ug total RNA was used for first strand cDNA synthesis using Superscript III First-Strand synthesis kit (Thermo Fisher Scientific). To evaluate relative expression levels of mRNAs, we performed qRT-PCR with the Power SYBR Green PCR master mix (Life Technologies, Carlsbad, CA) normalized to GAPDH. Detailed primer information is shown in Supplemental Table 1. We carried out real time PCR reaction and analyses in 384-well optical reaction plates using the CFX384 instrument (Bio-Rad, Hercules, CA).