Matt:LabNotes/2015-7-8

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iNGN Bipolar Neurons RNA FISH[edit]

  • 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[edit]

  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[edit]

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[edit]

  • Sample 1 labeled: "RNAFISH1 7.9.2015"
    • CUX2_odd-488 and CUX2_even-594 in Sebastian's neurons
  • Sample 2 labeled: "RNAFISH2 7.9.2015"
    • CUX2-488 only in Alex's neurons
  • Sample 3 labeled: "RNAFISH3 7.9.2015"
    • CUX2_odd-488 and CUX2_even-594 in Alex's neurons
  • Sample 4 labeled: "RNAFISH4 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 (~22hrs)
  8. Prepare 25ml Wash Buffer
  9. Warm up 16ml Wash Buffer and 8ml Wash Buffer with DAPI to 37C
  10. Rinse with 2ml Wash Buffer
  11. Add 2ml Wash Buffer and incubate 30min at 37C
  12. Replace with 2ml Wash Buffer with DAPI and incubate 30min at 30C
  13. Wash with 2X SSC (warmed to 30C) twice
  14. Add 2X SSC

Buffer Prep[edit]

  • 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

Imaging Results[edit]

  • RNAFISH1: Could see faint signal in 488 channel but nothing specific in 594 channel
    • Fainter signal in 488 compared to Third Try w/ cooled CCD camera is expected since only 24probes versus 48probes
    • Failure to see bright distinct spots in 594 channel but instead see bright regions of cells that correspond to dense dark areas in phase channel
      • My guess is that dye-probes or maybe free dyes got trapped?
  • RNAFISH2: Few spots in 488 representing CUX2 transcripts in Alex's neurons
    • I believe these neurons express less CUX2 than Sebastian's (based on FPKM~2 in Alex's RNA-Seq data and Sebastian claiming CUX2 is "highly expressed" in his neurons)
  • RNAFISH3: Results similar to RNAFISH1
  • RNAFISH4: CUX2 spots are detected but SNAP25 (which should have high expression) is not detected
    • The Alexa594 channel (SNAP25) shows the same bright regions of cells as the other dishes with 594 channel
      • This suggests the bright regions is not due to probe sequence but property of the dye
  • Conclusion:
    • Only CUX2-488 probes have worked and there is less CUX2 in Alex's iNGN bipolar neurons than Sebastian's motor neurons
    • CUX2-594 and SNAP25-594 both exhibited similar peculiarities and suggests the issue lies with Alexa594 dye or Alexa594 dye-coupling (See dye:probe ratio from dye-coupling results)
    • Need to call LifeTech ARES or Biosearch Stellaris customer support for advice on dye-coupling and bright regions
    • Next experiment should try switching probe and dye combinations (CUX2-488 and SNAP25-594)

Images[edit]

RNAFISH1[edit]

  • CUX2-488

File:4 w2Alexa488-Matt.jpg

  • CUX2-594

File:4 w1Alexa594-Matt.jpg

RNAFISH2[edit]

  • CUX2-488

File:RNAFISH2 20150709 4 w2Alexa488-Matt.jpg

RNAFISH3[edit]

  • CUX2-488

File:RNAFISH3 20150709 8 w2Alexa488-Matt.jpg

  • CUX2-594

File:RNAFISH3 20150709 8 w1Alexa594-Matt.jpg

  • Phase

File:RNAFISH3 20150709 8 w4Phase-WPZ.jpg

RNAFISH4[edit]

  • CUX2-488

File:RNAFISH4 20150709 9 w2Alexa488-Matt.jpg

  • SNAP25-594

File:RNAFISH4 20150709 9 w1Alexa594-Matt.jpg

  • Phase

File:RNAFISH4 20150709 9 w4Phase-WPZ.jpg

Settings[edit]

  • 7/10/2015
  • Dr. Bern's Zeiss microscope SERF 295
    • Light source: X-Cite 120LED
    • Objective: 100X NA=1.4 oil
    • Camera: QuantEM 512SC EMCCD Camera
    • Cubes: Color (Ex filter - Dichroic mirror - Em filter)
      • DAPI - (350/50 - 400 - 460/50)
        • 5% intensity 25msec exposure
      • FITC (Alexa488)- (480/30 - 510 - 535/40)
        • 100% intensity 5sec exposure
      • dsRED (Alexa594) - (545/30 - 570 - 628/52)
        • 20% intensity 150msec exposure

StarSearch[edit]