Deadenylation Assay Protocol
These assays are used to assess the rate and pattern of deadenylation of a radiolabeled RNA substrate. A single reaction is set up for each substrate and samples are taken at various time points during the experiment. The RNA substrate is recovered from each sample and run on a denaturing gel. Results are visualized by phosphorimager.
Reaction mix (assemble on ice):
16.0µl extract (protein concentration should be ~4-10µg/µl)
2.0µl poly(A) (500ng/µl, but should be titered for a new extract)
6.5µl 10% PVA (excludes H2O effectively increasing protein concentration)
2.0µl PC/ATP (ATP regeneration system)
2.0µl RNA substrate (50-200000 cpm/µl)
If you need four time points (e.g. 0, 10, 20, 30 min) take 6µl per time point.
Each 6µl sample should be added to 400µl HSCB containing 2µl tRNA.
Samples can then be stored at room temp for the duration of the experiment, or processed immediately.
Add 400µl phenol chloroform. Vortex. Spin 3 min.
Take the aqueous phase to a fresh tube containing 1ml ethanol.
Mix and precipitate at -80 ˚C for 10min.
Spin 10min. Wash pellet in 70% ethanol, and dry. Pellet should be clearly visible due to tRNA.
Resuspend in 8µl RNA loading dye, vortex and load on a pre-run 5% denaturing gel.
Run the gel far enough to separate the adenylated and deadenylated forms of the substrate.
Dry the gel and expose to phosphorimager screen for 1hr-overnight.