Daniel:Protocols/RNASort: Difference between revisions

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| width="127" | Volume in 10 mL
| width="180" | Volume in 10 mL


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Revision as of 16:32, 1 April 2015

RNA FISH Based Cell Sorting

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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

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

Buffer Prep

RPHB

Reagent Stock Concentration Final Concentration Dilution Volume in 10 mL
SSC Buffer 20X 2X 1:10 1 mL
Ammonium Sulfate NA, powder 2.1 M NA 2.775g
EDTA 0.5 M 10 mM 1:50 200 uL
E. coli tRNA 20 mg/mL 1 mg/mL 1:20 500 uL
BSA 50 mg/mL 500 ug/mL 1:100 100 uL
Formamide 100% 25% 1:04 2.5 mL
nfH2O - - - Fill to 10mL (~5 mL)

Sorting Buffer

Reagent Stock Concentration Final Concentration Dilution Volume in 10 mL
SSC Buffer 20X 2X 1:10 1 mL
Ammonium Sulfate NA, powder 1.5 M NA 1.982 g
EDTA 0.5 M 5 mM 1:100 100 uL
nfH2O - - - Fill to 10mL (~9 mL)

Reverse Cross-Linking Buffer

Reagent Stock Concentration Final Concentration Dilution Volume in 10 mL
NaCl NA, powder 100 mM - 58.4 mg
Tris 1 M 10 mM 1:100 100 uL
EDTA 0.5 M 1 mM EDTA 1:500 20 uL
SDS 10% w/v 0.5% (w/v) 1:20 500 uL
Proteinase K 20 mg/mL 500 ug/mL 1:40 250 uL
nfH2O - - - Fill to 10 mL (~9 mL)