Daniel:Notebook/RNAFISH/2015-2-5

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Revision as of 23:45, 5 February 2015 by >Djacobse (→‎Hybridization)
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Cell Line RNA FISH[edit]

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Slide Preparation[edit]

  1. Prepare a cell suspension of 500 000-2 000 000 cells/mL in culture media
  2. Add 20 uL of cell suspension per dish to the cener; add 80 uL culture media
  3. Allow cells to adhere for 6 hours at 37 C
  4. Rinse slides briefly in 1X PBS
  5. Fix slides for 5 minutes in 4% (v/v) paraformaldehyde in 1X PBS
  6. Wash 2x in 2xSSPE for 5 minutes each

Hybridization[edit]

Sample Matrix

Sample uL FOXP2-488 uL GAD1-594 uL hybridization buffer uL formamide uL Total
A 1 1 20.5 2.5 25
B 2 2 18.5 2.5 25
  1. Permeabilize by leaving at room temperature for 30 minutes in 0.6% Triton X-100/2xSSPEdepc
  2. Wash 2x for 5 minutes in 2X SSPE
  3. Dehydrate in 30, 50, 70, 100, 100 ethanols 2 mins ea.
    1. Make solutions from absolute ethanol and depc/H20*.
  4. Dry in oven at 50-65C.
  5. Prehybridize slides ~ 1 hour+ at 70C with prehybridization solution
  6. Determine probe concentration (usually 2-4ng/ul) and denature probe at 90C for 10-15 minutes.
  7. Drop temp to 65C
  8. Spin briefly. Do not open the tube when it is at 90C-probe loss can occur.
  9. Add about 50-100 ng of probe to each slide in appropriate buffer; should be ~25 uL
  10. Seal coverslip with rubber cement, wait ~5 minutes for cement to dry
  11. Hybridize overnight appropriate 37C
  • 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