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)

----
28.5µl

 

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.
i.e. larger RNAs should be run longer.

Dry the gel and expose to phosphorimager screen for 1hr-overnight.

Notes:

  • Always keep a note of the extract used (date) and any variations from the above protocol.
  • It’s important to remove the salt from the RNA before running so be sure to remove all the ethanol before drying.
  • If the interphase during phenol/chloroform extraction is very large then try using phenol instead.
  • Be sure to do appropriate controls every time – usually Gem A60 is good to get a general deadenylation rate for a specific extract.
  • If you need to compare two RNAs that are very different sizes then you can load the larger one on the gel earlier than the smaller one.
  • Remember you’re working with radio-active RNA – monitor for contamination and use RNAse free solutions.