Daniel:Notebook/RNAFISH/2014-12-15
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Cell Line RNA FISH 2
Since I have two more samples and not enough time to really do much else, I'm going to retry the cell line FISH.
Hybridization
Sample Matrix
Sample | Cell Line | uL GAD1-488 (ng) | uL CUX2-594 (ng) |
A2 | U87-MG | 3 (85) | 4 (86) |
B2 | BE2-C | 3 (85) | 4 (86) |
- Permeabilize by leaving at room temperature for 30 minutes in 2X SSCT+50% formamide
- Dehydrate in 30, 50, 70, 100, 100 ethanols 2 mins ea.
- Make solutions from absolute ethanol and depc/H20*.
- Dry in oven at 50-65C.
- Prehybridize slides ~ 1 hour+ at 70C with prehybridization solution
- Determine probe concentration (usually 2-4ng/ul) and denature probe at 90C for 10-15 minutes.
- Drop temp to 65C
- Spin briefly. Do not open the tube when it is at 90C-probe loss can occur.
- Add about 50-100 ng of probe to each slide in appropriate buffer; should be ~25 uL
- Seal coverslip with rubber cement, wait ~5 minutes for cement to dry
- Hybridize overnight appropriate temperature
- VARIABLE--shorter probes may want much lower temp; Hybridization temp may need to be determined empirically (=many trials…….)
- important to keep slides in a humid environment
- Prehybridization solution:
- 50% deionized formamide
- 50% 2xSSC pH 7.0
- Add dextran sulfate to 1%
- Hybridization solution (Arjun Raj)
- 2X SSC
- 10% Formamide
- 10% Dextran Sulfate
- 0.02% RNAse-free BSA
- 50 μg E.coli tRNA