Daniel:Protocols/RNASort

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Revision as of 18:30, 31 March 2015 by >Djacobse (Created page with "=RNA FISH Based Cell Sorting= Back to Main [http://www.nature.com/nmeth/journal/v11/n5/pdf/nmeth.2910.pdf Original paper by Klemm et al] The goal of thi...")
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RNA FISH Based Cell Sorting

Back to Main

Original paper by Klemm et al

The goal of this protocol is to be able to use RNA FISH when doing cell sorting, which can be a potentially more valuable way of cell sorting than using antibody staining, as it provides greater discrimination prospects.

Protocol

NOTE: The original protocol (from the paper) is rather vague and several steps may require troubleshooting if my guesses as to their meaning are not correct.

RNA FISH

  1. Fix cells in 4% paraformaldehyde for 5 min
  2. Centrifuge cells at 1000g for 5 minutes
  3. Wash cells with 70% EtOH
  4. Ethanol permeabilization- 70% EtOH overnight at 4C
  5. Resuspend cells in RPHBwith labeled probes (0.5-1ng/uL)
    1. Incubate for 12 hrs at 30C
  6. Add equal volume of wash buffer and mix thoroughly
  7. Pellet by centrifugation (1000g for 5 min)
  8. Resuspend cells in wash buffer for 30 minutes at 30C
  9. Pellet by centrifugation and resuspend cells in wash buffer for 30 minutes at 30C
  10. Pellet cells and resuspend in flow buffer at 4C in preparation for sorting

Flow Cytometry, FACS, and RNA Extraction

  1. Sort by FACS into 4C sorting buffer
  2. Cell size was controlled by costaining with Hoeschst 33342 and selecting for diploid DNA content
  3. Pellet FACS sorted cells using 1000g for 5 minutes and resuspend in reverse cross-linking buffer at 50C for 1 hr
  4. Isolate total RNA via phenol-chloroform or other