Quantifying mRNA Stability by qRT-PCR

RNA stability assay was performed by qRT-PCR as previously reported (76 (link), 81 (link)). Briefly, cells were treated with actinomycin D (10 μg/ml, Fisher) to block transcription, and RNAs were isolated at various time points after actinomycin D treatment. qRT-PCR was then performed using 25 ng of template cDNA for each mRNA gene of interest. Each sample was run in triplicate. The relative abundance of each mRNA was calculated using the ΔΔC_T method and normalized to Gapdh. The relative amount of mRNA at 0 h following actinomycin D treatment was arbitrarily set to 1. Curve fittings of the resultant data were performed using Microsoft Excel and the half-lives of the RNAs calculated.