Daniel:Protocols/RNASort: Difference between revisions
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>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...") |
>Djacobse |
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#Pellet FACS sorted cells using 1000g for 5 minutes and resuspend in reverse cross-linking buffer at 50C for 1 hr | #Pellet FACS sorted cells using 1000g for 5 minutes and resuspend in reverse cross-linking buffer at 50C for 1 hr | ||
#Isolate total RNA via phenol-chloroform or other | #Isolate total RNA via phenol-chloroform or other | ||
===Buffers=== | |||
*'''RPHB''' | |||
**300 mM NaCl | |||
**30 mM sodium citrate | |||
**2.1M ammonium sulfate | |||
**10 mM EDTA | |||
**1 mg/mL E. coli tRNA | |||
**500 ug/mL BSA | |||
**25% v/v formamide | |||
**pH 5.2 | |||
*'''Wash Buffer''' | |||
**25% v/v formamide | |||
**2X SSC buffer | |||
*'''Flow Buffer''' | |||
**2X SSC | |||
*'''Sorting Buffer''' | |||
**200mM NaCl | |||
**20 mM sodium citrate | |||
**1.5M ammonium sulfate | |||
**5 mM EDTA | |||
**2X SSC | |||
**pH 5.2 | |||
*'''Reverse Cross-Linking Buffer''' | |||
**100mM NaCl | |||
**10mM Tris | |||
**1 mM EDTA | |||
**0.5% SDS (v/v) | |||
**500 ug/mL proteinase K |
Revision as of 20:39, 31 March 2015
RNA FISH Based Cell Sorting
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
- Fix cells in 4% paraformaldehyde for 5 min
- Centrifuge cells at 1000g for 5 minutes
- Wash cells with 70% EtOH
- Ethanol permeabilization- 70% EtOH overnight at 4C
- Resuspend cells in RPHBwith labeled probes (0.5-1ng/uL)
- Incubate for 12 hrs at 30C
- Add equal volume of wash buffer and mix thoroughly
- Pellet by centrifugation (1000g for 5 min)
- Resuspend cells in wash buffer for 30 minutes at 30C
- Pellet by centrifugation and resuspend cells in wash buffer for 30 minutes at 30C
- Pellet cells and resuspend in flow buffer at 4C in preparation for sorting
Flow Cytometry, FACS, and RNA Extraction
- Sort by FACS into 4C sorting buffer
- Cell size was controlled by costaining with Hoeschst 33342 and selecting for diploid DNA content
- Pellet FACS sorted cells using 1000g for 5 minutes and resuspend in reverse cross-linking buffer at 50C for 1 hr
- Isolate total RNA via phenol-chloroform or other
Buffers
- RPHB
- 300 mM NaCl
- 30 mM sodium citrate
- 2.1M ammonium sulfate
- 10 mM EDTA
- 1 mg/mL E. coli tRNA
- 500 ug/mL BSA
- 25% v/v formamide
- pH 5.2
- Wash Buffer
- 25% v/v formamide
- 2X SSC buffer
- Flow Buffer
- 2X SSC
- Sorting Buffer
- 200mM NaCl
- 20 mM sodium citrate
- 1.5M ammonium sulfate
- 5 mM EDTA
- 2X SSC
- pH 5.2
- Reverse Cross-Linking Buffer
- 100mM NaCl
- 10mM Tris
- 1 mM EDTA
- 0.5% SDS (v/v)
- 500 ug/mL proteinase K