Daniel:Notebook/RNAFISH/2014-12-9
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Cell Line RNA FISH
Dye Labeling
Since I am planning on doing RNA FISH using CUX2 and GAD1, and since I would like to use Alexa 488 and 594, the best dyes I think, I need to do dye labeling. I should be able to use the Alexa dyes, since these are the probes I ordered from IDT.
- Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
- Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
- Denature samples for 5 minutes at 95C, then snap cool using ice box
- Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
- dye cannot be saved for later use. Use immediately!
- Add 1 uL dye to sample
- Incubate in the dark for 1 hour
- Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
- Recommend column purification after incubation-Centri-Sep Column
Labeling Results
Sample | ng/uL ssDNA | pmol/uL dye | pmol/uL ssDNA | dye:probe | bp:dye |
GAD1-488 | 28.3 | 5 | 4.3 | 1.2 | 17 |
CUX2-594 | 21.4 | 4 | 3.2 | 1.2 | 16 |
Hybridization
- 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