a, RT-PCR for pri-let-7g transcript (as described in ref. (7)) during ES differentiation to embryoid bodies. Actin serves as control. b, Northern blot showing post-transcriptional induction of mature let-7g during embryoid body formation 5S rRNA serves as loading control. c, in vitro pri-miRNA processing reaction using radiolabeled pri-let-7g as substrate. Pri-miRNA was pre-incubated with various amounts of P19 cell extract or mouse embryonic fibroblast (MEF) extract prior to processing reaction with Flag-Drosha immunoprecipitate, as described in Methods. The ratio of pre-miRNA to pri-miRNA was quantitated by densitometry and values were normalized to the Microprocessor only lane. d, qPCR analysis of gene expression during embryoid body formation of a feeder-free mouse ES line (J1 ES) . Top Panel: Pri-let-7g and mature let-7g; Middle Panel: Lin-28; Bottom Panel: pluripotency factors Oct-4 and Nanog.