Genome-wide miRNA turnover profile in various cell types after transcription inhibition. Genome-wide miRNA turnover profile in various cell types after transcription inhibition

We measured miRNA turnover rates in 8 mammalian cell types post transcription inhibitors ActinomycinD (ActD) or 5,6-Dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) treatment with miRNA expression profiling, generating 218 expression profiles on 528 endogenous miRNAs. Overall design: Cells were plated in six-well plates at around 200,000 cells/well. A media change was performed one day after plating. ActD and DRB treatment were started after confluency (around two days after plating) for 0, 2, 4, 6, 12, and 24 hours post treatment with biological duplicate (ActD treatment) or biological triplicate (DRB treatment). The concentration of ActD and DRB for different cell lines were titrated to achieve rapid c-myc down-regulation after 2 hours of ActD and DRB treatment. After treatment at indicated time points, cells were washed with PBS once and lysed in TriZol reagent immediately (Invitrogen). RNA extraction was performed following the manufacturer’s protocol. miRNA profiling was performed using a Luminex bead-based method.