Daniel:Notebook/RNAFISH/2014-11-24: Difference between revisions

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#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.   
#Dehydrate in 30, 50, 70, 100, 100 ethanols 2 mins ea.   
##Make solutions from absolute ethanol and depc/H20*.
##Make solutions from absolute ethanol and depc/H20*.

Revision as of 19:20, 24 November 2014

RNA FISH-Cell Lines

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Hybridization

Today I'm going to perform hybridization on the cell lines I fixed Saturday. I'll be using RBFOX3 (NeuN) and FOXP2 as my genes of choice.

Sample Matrix

Sample Cell Line uL RBFOX3-488 (ng) uL FOXP2-594 (ng)
A1 BE2-C 2 (110) 2 (120)
B1 T98-G 2 (110) 2 (120)
C1 U-87MG 2 (110) 2 (120)
D1 NCI h1975 2 (110) 2 (120)
  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