Daniel:Notebook/RNAFISH/2014-8-31

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Revision as of 15:59, 3 September 2014 by >Djacobse
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Trial Run (Started 08/29/2014)

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Dye Labeling

Sample Matrix

Sample GAD1 FOXP2 RBFOX3 CUX2
Dye Alexa-488 Alexa-546 Alexa-594 Alexa-647
ng/uL 512 558 568 604
uL added 10 10 10 10
ug added 5.1 5.6 5.7 6.0
  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

Sample ng/uL ssDNA pmol/uL dye pmol/uL ssDNA dye:probe bp:dye
GAD1-488 55.7 9.2 8.4 1.1 18
FOXP2-546 67.7 13.7 10.3 1.3 15
RBFOX3-594 68.3 13.8 10.3 1.3 15
CUX2-647 59.8 12.3 9.1 1.4 15

Great labeling results, and each sample has ~50 uL to work with.

Pretreatment

Sample Matrix

  uL GAD1-488 (pmol) uL FOXP2-546 (pmol) uL RBFOX3-594 (pmol) uL CUX2-647 (pmol)
Sample A 25 (211) 0 25 (258.7) 0
Sample B 0 25 (256.4) 0 25 (226.5)

Sample A is the sample closer to the label, sample B is the sample further from the label.

  1. Remove slides from 4C, let them thaw and air-dry at RT for 30–60 min
  2. Re-hydrate sections in 10 mM sodium citrate buffer for 5 min
  3. Transfer slides into a Coplin jar with prewarmed (80°C) 10 mM sodium citrate buffer in a water bath and incubate there for 5–25 min
  4. Remove the Coplin jar from the water bath and let it cool down at RT
  5. Incubate the slides in 2× SSC for 5 min
  6. Transfer slides into 50% formamide in 2× SSC and incubate for at least 1 h

Hybridization

  1. Remove slide from the formamide and wipe the liquid from the back of the slide and from the face side of the slide
    1. Avoid marked section area
  2. Prepare glass hybridization chambers as follows:
    1. Roll a small piece of mounting putty (Blu Tack) into a snake.
    2. Wrap the mounting putty around tissue section, covering 3 sides
    3. Size of the chamber depends on a section size: chamber should fit to the section size as closely as possible in order to save DNA probe
  3. Place coverslip over putty, press gently to seal the coverslip to the putty
  4. Load the probe under chamber from its open side so that probe fills in the whole space above the section
  5. Seal the chamber with rubber cement
    1. It is important that rubber cement covers the entire chamber periphery or even the whole chamber
  6. Let the rubber cement dry at RT
    1. From this step, protect slide from light
  7. Place slide on a hot-block (or water bath) with temperature 45°C for 2 h in order to allow infiltration of the section with the probe
  8. Place slide on a hot-block at 80°C for 5 min for the probe and section denaturation of DNA
  9. Hybridize slide for 3 days at 37°C in a waterbath or temperature controlled oven; Continued 9/3/2014