On the optimal design of metabolic RNA labeling experiments
Fig 5
Purification of labeled and unlabeled RNA fractions.
MCF-7 cells were pulse labeled with 4sU for up to eight hr as indicated. Total RNA was spiked with in vitro transcribed 4sU-labeled FLuc and unlabeled RLuc, biotinylated with MTSEA-biotin and subjected to streptavidin purification. (n = 3). A: Dot blot-based detection of biotinylation with streptavidin-HRP in input and flow through of streptavidin purification. B: The amount of RNA enriched by the streptavidin purification was determined by absorption measurement. C: In vitro transcribed spike in RNAs 4sU-labeled FLuc and unlabeled RLuc in the flow through and biotin-enriched fraction were measured by RT-qPCR analysis and normalized to a standard curve given in S3 Fig.