Daniel:Notebook/RNAFISH/2014-12-9

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Revision as of 21:11, 9 December 2014 by >Djacobse (→‎Cell Line RNA FISH)
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Cell Line RNA FISH

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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.

  1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
  2. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
  3. Denature samples for 5 minutes at 95C, then snap cool using ice box
  4. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
    1. dye cannot be saved for later use. Use immediately!
  5. Add 1 uL dye to sample
  6. Incubate in the dark for 1 hour
  7. Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
  8. 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

  1. Permeabilize by leaving at room temperature for 30 minutes in 2X SSCT+50% formamide
  2. Dehydrate in 30, 50, 70, 100, 100 ethanols 2 mins ea.
    1. Make solutions from absolute ethanol and depc/H20*.
  3. Dry in oven at 50-65C.
  4. Prehybridize slides ~ 1 hour+ at 70C with prehybridization solution
  5. Determine probe concentration (usually 2-4ng/ul) and denature probe at 90C for 10-15 minutes.
  6. Drop temp to 65C
  7. Spin briefly. Do not open the tube when it is at 90C-probe loss can occur.
  8. Add about 50-100 ng of probe to each slide in appropriate buffer; should be ~25 uL
  9. Seal coverslip with rubber cement, wait ~5 minutes for cement to dry
  10. Hybridize overnight appropriate temperature
    1. VARIABLE--shorter probes may want much lower temp; Hybridization temp may need to be determined empirically (=many trials…….)
    2. 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