Matt:LabNotes/2015-7-8
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iNGN Bipolar Neurons RNA FISH[edit]
- First Try
- Second Try
- Probe dye coupling
- Third Try with cooled CCD camera
- Finally saw some signal using CUX2-Alexa488 48 probes with Sebastian's motor neurons
- Camera did not have DAPI filters so did not use DAPI
- Only used one probe set per sample
- Samples are iNGN Bipolar Neuron's from Alex at Harvard
- Fixed 6/30/2015 and stored in 70% EtOH at 4C for ~9days before RNA FISH
- RNA-Seq FPKM data
- RNA FISH 48 Probes available
- CUX2, SLC6A1, ADARB2, SATB2, KIT, SNAP25
- ADARB2 not in DARTFISH CA12kNov2014_V4
- CUX2, SLC6A1, ADARB2, SATB2, KIT, SNAP25
- 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]
- Oligos already resuspended in 15 uL nuclase-free H2O (1 mM)
- Take 2 uL from each oligo well and combine them in a single 1.5 mL tube (total concentration 1 mM)
- Store the remaining oligos at -20C
- 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
- 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
- Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature
- Add 5 uL DNA (~1ug/ul) and 3 uL of sodium bicarbonate buffer
- Denature samples for 5 minutes at 95C, then snap cool using ice box
- 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!
- Add 8 uL sample to dye tube
- Incubate in the dark for 1 hour
- Add 10 uL 3M NaOAc and 80 uL 1xTE to sample
- Centri-Sep column purification after incubation
- Use 1X TE Buffer
- No vacufuge
- Disturbed gel bed in CUX2_odd-488
- Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight
- Centrifuge at 4 C for 30 minutes (14,000 rpm)
- Remove supernatant and add 750 uL chilled 80% EtOH
- Centrifuge 5 minutes at 4 C
- Dry the pellet in the hood
- Resuspend pellet in 12 uL TE
- 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
- Prepare 10ml Wash Buffer and let sit at RT
- Thaw 500ul aliquot Hybridization Buffer and warm up to 37C
- Aliquots made by Dan
- Prepare hybridization reaction
- Hybridization Buffer 100ul
- Probe 5ul for each dye-probe (final volume will be 105ul or 110ul)
- Wash the cells with 1ml of PBS (RNase free) 2 times
- Add 2ml Wash Buffer and let sit 10min at RT
- Aspirate
- Add 105ul or 110ul hybridization solution to each sample and incubate overnight at 37C (~22hrs)
- Prepare 25ml Wash Buffer
- Warm up 16ml Wash Buffer and 8ml Wash Buffer with DAPI to 37C
- Rinse with 2ml Wash Buffer
- Add 2ml Wash Buffer and incubate 30min at 37C
- Replace with 2ml Wash Buffer with DAPI and incubate 30min at 30C
- Wash with 2X SSC (warmed to 30C) twice
- 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
- The Alexa594 channel (SNAP25) shows the same bright regions of cells as the other dishes with 594 channel
- 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
- CUX2-594
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
- DAPI - (350/50 - 400 - 460/50)