Daniel:Notebook/RNAFISH/2014-10-11

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ABP Trial Run (Started 10/10/2014)[edit]

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Dye Labeling-ARES[edit]

I need to make the appropriate probes, so I'll be labeling with the appropriate ARES dyes.

Sample FOXP2 RBFOX3
Dye Alexa-594 Alexa-488
ng/uL 558 568
uL added 10 10
ug added 5.6 5.7
  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 2 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. Centri Sep purification after incubation

Labeling Results[edit]

Sample ng/uL ssDNA pmol/uL dye pmol/uL ssDNA dye:probe bp:dye
FOXP2-594 61.3 11.9 9.3 1.3 16
RBFOX3-488 54.9 9 8.3 1.1 18

As usual wit ARES, looks great.

Hybridization[edit]

  1. Prehybridize slides ~ 1 hour+ at 70C with prehybridization solution
  2. Determine probe concentration (usually 2-4ng/ul) and denature probe at 90C for 10-15 minutes.
  3. Drop temp to 65C
  4. Spin briefly. Do not open the tube when it is at 90C-probe loss can occur.
  5. Add about 50-100 ng of probe to each slide in appropriate buffer; should be ~25 uL
  6. Seal coverslip with rubber cement, wait ~5 minutes for cement to dry
  7. 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

Buffers[edit]

  • Prehybridization solution:
    • 50% deionized formamide
    • 50% 2xSSC pH 7.0
    • Add dextran sulfate to 1%
  • Hybridization solution A-Long Cai
    • 2X SSC
    • 20% Formamide
    • 10% Dextran Sulfate
  • Hybridization solution B-Arjun Raj
    • 2X SSC
    • 10% Formamide
    • 10% Dextran Sulfate
    • 0.02% RNAse-free BSA
    • 50 μg E.coli tRNA