Daniel:Protocols/RNASort
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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
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 (~ mL) |