Matt:LabNotes/2015-7-8

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Revision as of 00:06, 10 July 2015 by >Mzcai (→‎RNA FISH Procedure)
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iNGN Bipolar Neurons RNA FISH

  • RNA FISH 48 Probes available
    • CUX2, SLC6A1, ADARB2, SATB2, KIT, SNAP25
      • ADARB2 not in DARTFISH CA12kNov2014_V4
  • SNAP25 has highest FPKM value
  • CUX2 is low-medium expression but use as positive control because it was the only one that worked previously
    • One sample exact same as last time CUX2-Alexa488 with no DAPI
    • One sample with odd CUX2-Alexa488 and even CUX2-Alexa594 to check specificity (should colocalize or else non-specific binding)
    • One sample with CUX2-Alexa488 and SNAP25-Alexa 594

Combining CUX2_odd and CUX2_even

  1. Oligos already resuspended in 15 uL nuclase-free H2O (1 mM)
  2. Take 2 uL from each oligo well and combine them in a single 1.5 mL tube (total concentration 1 mM)
    1. Store the remaining oligos at -20C
  3. Add 230 uL nuclase-free, diluting 10:1, for final concentration of 100 uM
    • Technically closer to 5:1 dilution but final result is closer to 10:1 because starting concentration is actually lower...
    • Goal is to get 1ug/ul DNA concentration
  4. Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy
' measured (ng/ul) normalized (ng/ul) Average Stdev
CUX2_odd 1:1 1251.5 1251.5
CUX2_odd 1:5 247 1235
CUX2_odd 1:10 117.4 1174 1220 40.8
CUX2_even 1:1 1251.8 1251.8
CUX2_even 1:5 235.9 1179.5
CUX2_even 1:10 119.1 1191 1207 38.9
  • Added 55ul H2O to each (275ul) to make 330ul of 1000ng/ul

Dye Coupling

Dan's best practice dye coupling protocol
Generic dye coupling protocol

CUX2-488 CUX2_odd-488 CUX2_even-594 SNAP25-594

  1. Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature
  2. Add 5 uL DNA (~1ug/ul) 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 DMSO; do multiple cycles of vortex and spin down during 5 minutes of denaturing
    • Dye cannot be saved for later use. Use immediately!
  5. Add 8 uL sample to dye tube
  6. Incubate in the dark for 1 hour
  7. Add 10 uL 3M NaOAc and 80 uL 1xTE to sample
  8. Centri-Sep column purification after incubation
    • Use 1X TE Buffer
    • No vacufuge
    • Disturbed gel bed in CUX2_odd-488
  9. Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight
  10. Centrifuge at 4 C for 30 minutes (14,000 rpm)
  11. Remove supernatant and add 750 uL chilled 80% EtOH
  12. Centrifuge 5 minutes at 4 C
  13. Dry the pellet in the hood
  14. Resuspend pellet in 12 uL TE
  15. Check dye:probe ratio with Nanodrop
    • Use 324.5 g/mol/bp as molar mass of ssDNA (6490 g/mol of 20-mer)
    • Starting DNA mass = 5ul * 1ug/ul
Probe ssDNA (ng/ul) 488 dye (pmol/ul) 594 dye (pmol/ul) dye:probe ratio ssDNA mass (ug) ssDNA start mass (ug) ssDNA Yield (%)
CUX2-488 197.2 33.1 0.5 1.09 2.37 5 47.3
CUX2_odd-488 244 40.6 0.6 1.08 2.93 5 58.6
CUX2_even-594 295.9 -0.8 57.3 1.26 3.55 5 71
SNAP25-594 227.4 -0.6 47.5 1.36 2.73 5 54.6

RNA FISH Procedure

  • Sample 1 labeled: "RNAFISH1 7.9.2015"
    • CUX2-488 only in Alex's neurons
  • Sample 2 labeled: "RNAFISH2 7.9.2015"
    • CUX2_odd-488 and CUX2_even-594 in Alex's neurons
  • Sample 3 labeled: "RNAFISH3 7.9.2015"
    • CUX2-488 and SNAP25-594 in Alex's neurons
  1. Prepare 10ml Wash Buffer and let sit at RT
  2. Thaw 500ul aliquot Hybridization Buffer and warm up to 37C
    • Aliquots made by Dan
  3. Prepare hybridization reaction
    • Hybridization Buffer 100ul
    • Probe 5ul for each dye-probe (final volume will be 105ul or 110ul)
  4. Wash the cells with 1ml of PBS (RNase free) 2 times
  5. Add 2ml Wash Buffer and let sit 10min at RT
  6. Aspirate
  7. Add 105ul or 110ul hybridization solution to each sample and incubate overnight at 37C (~23hrs)

Buffer Prep

  • Wash Buffer
    • 20X SSC 5mL
    • Formamide 5mL
    • RNase free H2O 40ml
  • Wash Buffer with DAPI
    • Wash Buffer 10ml
    • DAPI (100ug/ml) 50ul
      • Stored in brown opaque tube in 4C
  • Hybridization Buffer
    • RNAse free water 5.3 mL
    • SSC 20X 1 mL
    • Dextran sulfate 2 mL
    • Formamide 1 mL
    • E coli tRNA 500 uL
    • RVC 200 mM (warm to 37) 100 uL
    • BSA 50 mg/mL 40 uL