Comparison of approaches for measuring mRNA decay kinetics. (A) mRNA decay measurements using Roadblock-qPCR and transcriptional shutoff with ActD on endogenous mRNAs. RNA was isolated from HEK-293T cells treated with either 2 µg/mL Actinomycin D or 400 µM 4sU for the indicated times. mRNA from 4sU-exposed cells was treated with NEM before reverse transcription. Levels of the indicated mRNAs at each timepoint were determined by qPCR. Error bars are SD, n = 3. (B) Half-life calculations using Roadblock-qPCR and 4sU-biotin are similar. Roadblock-qPCR was performed on HEK-293T cells for 11 mRNAs using 2, 4 and 8 h timepoints of 4sU (400 µM) treatment. The calculated half-lives were compared to those obtained previously using affinity purification of biotinylated 4sU-containing mRNA (Friedel and Dolken 2009). Note: Correlation calculations for B-cell and combined data sets exclude SLC7A11, an extreme outlier. (C) Half-life calculations obtained from Roadblock-qPCR and TimeLapse-seq are similar. Half-lives calculated in B were compared to those reported previously with TimeLapse-seq on K562 cells (Schofield et al. 2018).
