RNA FISH Based Cell Sorting[edit]
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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[edit]
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[edit]
- 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 RPHB with 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[edit]
- 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[edit]
- 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
- 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[edit]
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[edit]
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[edit]
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)
|