Matt:LabNotes/2015-6-30
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Motor Neurons RNA FISH with cooled CCD[edit]
- Photometric QUANTEM Camera
- Cooled to -30C
- 100X objective
- Ex/Em Filters available
- Alexa488
- 495/519 (FITC equivalent)
- Filter 5/Filter 2
- Alexa564
- 556/573 (tetramethylrhodamine)
- No filters available
- Alexa594
- 590/617 (Texas Red)
- Filter 8/Filter 5
- Alexa647
- 647/665 (Cy5)
- Filter 4/Filter 6-7
- Second batch iPS derived motor neurons from Yeo Lab (Sebastian)
- Permeabilized/stored in 70% EtOH at 4C (2 weeks before RNA FISH experiment)
RNA FISH Procedure[edit]
- Using CUX2-488 KIT-594 KIT-647
- Sample 1 labeled: "RNAFISH1 6.30.2015"
- 488
- Sample 2 labeled: "RNAFISH2 6.30.2015"
- 594
- Sample 3 labeled: "RNAFISH3 6.30.2015"
- 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
- Hybridization Buffer 100ul
- Probe 5ul
- Wash the cells with 1ml of PBS (RNase free) 2 times
- Add 2ml Wash Buffer and let sit 10min at RT
- Aspirate
- Add 105ul hybridization solution to each sample and incubate overnight at 37C (~23hrs)
- Prepare 15ml Wash Buffer
- Warm up 12ml Wash Buffer and 3ml 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
Results[edit]
- Only CUX2 (488) probes worked
- My guess is CUX2 is most highly expressed in these cells
- To confirm next time I can use CUX2 probes labeled with 2 different dyes
- Odd probes labeled one color and even probes labeled the other, should see colocalization of 2 colors
Raw Images[edit]
- RNAFISH1_Pos1
- Exposure only 1sec vs 5sec for other images
File:2 w1Alexa488.jpg File:2 w2DIC.jpg
- RNAFISH1_Pos2
File:3 w1Alexa488.jpg File:3 w2DIC.jpg
- RNAFISH1_Pos3
File:5 w1Alexa488.jpg File:5 w2DIC.jpg
- RNAFISH1_Pos4
File:7 w1Alexa488.jpg File:7 w2DIC.jpg
- RNAFISH3_Pos1 with 488 excitation (use exact same microscope settings as RNAFISH1 for Negative Control)
File:Dish3 5 w1Alexa488.jpg File:Dish3 5 w2DIC.jpg
- RNAFISH3_Pos2 with 488 excitation (use exact same microscope settings as RNAFISH1 for Negative Control)
File:Dish3 6 w1Alexa488.jpg File:Dish3 6 w2DIC.jpg
StarSearch[edit]
- 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"
- RNAFISH1_Pos2
File:3 w1Alexa488 StarSearch.png
- RNAFISH1_Pos3
File:5 w1Alexa488 StarSearch.png
- RNAFISH1_Pos4
File:7 w1Alexa488 StarSearch.png
- RNAFISH3_Pos1 with 488 excitation (use exact same microscope settings as RNAFISH1 for Negative Control)
- Can't process image, something about StarSearch algorithm makes image almost all white/saturated
- RNAFISH3_Pos2 with 488 excitation (use exact same microscope settings as RNAFISH1 for Negative Control)
- Identified 6 spots but you can tell they're not real distinct spots