Matt:LabNotes/2015-6-18
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Motor Neurons RNA FISH
- Picked up second batch iPS derived motor neurons from Yeo Lab (Sebastian)
- Fixed in 4% paraformaldehyde (fresh) at 3:00p 6/16/2015
- RT for 15min
- Permeabilized/store in 70% EtOH at 4C (~48 hours before RNA FISH experiment)
- Fixed in 4% paraformaldehyde (fresh) at 3:00p 6/16/2015
RNA FISH Procedure
- Sample 1 labeled: "RNAFISH1 6.18.2015"
- 488 and 594
- Sample 2 labeled: "RNAFISH2 6.18.2015"
- 546 and 647
- 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 (2X volume)
- Hybridization Buffer 200ul
- Probe 10ul of each (5ul-488+5ul-594 and 5ul-546+5ul-647)
- Wash the cells with 1ml of PBS (RNase free) 2 times
- Add 1ml Wash Buffer and let sit 10min at RT
- Aspirate
- Add 110ul hybridization solution to each sample and incubate overnight at 37C (~18hrs)
- Warm up 6ml Wash Buffer and 2ml Wash Buffer with DAPI to 37C
- Rinse with 2ml Wash Buffer
- Add 1ml Wash Buffer and incubate 30min at 37C
- Replace with 1ml Wash Buffer with DAPI and incubate 30min at 37C
- Wash with 2X SSC (warmed to 30C) twice
- Add 2X SSC
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
Results
- Olympus Imaging 6/19/2015 (Stored in DARTFISH-RNAFISH folder on desktop)
- RNAFISH1: Alexa 488 (GFP filter) and Alexa 594 (TXRED filter)
- RNAFISH2: Alexa 546 (Cy3 filter) and Alexa 647 (Cy5 filter)
- Use StarSearch from Raj Lab
- Doesn't work in Chrome browser (use IE)
- Uses a histogram of pixel intensities and a threshold to determine how many "stars"
- Still see a lot more background than example images on Raj lab website
RNAFISH1
- Pos1 488-CUX2 (GFP)
- Most spots aren't in cell
File:RNAFISH1 Pos1 GFP StarSearch.png
- Pos1 594-KIT (TXRED)
- Probably not mRNA signal?
File:RNAFISH1 Pos1 TXRED StarSearch.png
- Pos2 488-CUX2 (GFP)
File:RNAFISH1 Pos2 GFP StarSearch.png
- Pos2 594-KIT (TXRED)
File:RNAFISH1 Pos2 TXRED StarSearch.png
RNAFISH2
- Pos1 546-CUX2 (Cy3)
- Pos1 647-KIT (Cy5)
- Pos2 546-CUX2 (Cy3)
- Pos2 647-KIT (Cy5)