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==sm RNA FISH== *Using [[Matt:LabNotes/2015-7-14 | KIT-488 CUX2-594]] **Expect to see very few to no signals in FITC channel due to few KIT mRNA **If TXRED channel shows CUX2 transcripts then dye:probe=1.3 is not the problem **If TXRED channel shows bright regions then probe sequences not the problem *Using [[Matt:LabNotes/2015-7-16 | SNAP25-488 CUX2-647]] **Expect to see many SNAP25 spots in FITC channel **Hopefully can also see a couple CUX2 spots in Cy5 channel ===Procedure=== *[[Daniel:Notebook/RNAFISH/2015-3-4 | Dan's best practice FISH protocol]] *[[Daniel:Protocols/TissueFISH#Linnarsson_Lab_Protocol | Linnarsson complete FISH protocol]] *Sample 1 labeled: "RNAFISH1 7.20.2015" **KIT-488 CUX2-594 *Sample 2 labeled: "RNAFISH2 7.20.2015" **SNAP25-488 CUX2-647 #Prepare 10ml Wash Buffer and let sit at RT #Thaw 300ul aliquot Hybridization Buffer and warm up to 37C #*Aliquots made by Dan #Prepare hybridization reaction #*Hybridization Buffer 100ul #*Probe 5ul + 5ul #Wash the cells with 1ml of PBS (RNase free) 2 times #Add 2ml Wash Buffer and let sit 10min at RT #Aspirate #Add 110ul hybridization solution to each sample and incubate overnight at 37C (~23hrs) #Prepare 12ml Wash Buffer #Warm up 8ml Wash Buffer and 4ml 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==== *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=== ====Zeiss Microscope in Huang Lab with Laser excitation==== *7/21/2015 *With help of Matt Walsh *Laser wavelengths: **488 **552? **642 *Exposure time: 100ms *Sample 1 - 488 (KIT) **Nothing to show, probably because gene expression is too low *Sample 1 - 594 (CUX2) **See combination of bright regions (guess is due to uncoupled dyes) and very few bright spots [[File:20150720_RNAFISH1_594.jpg]] *Sample 2 - 488 (SNAP25) **Lots of spots but also high background ***Hard to say significantly more than what I have seen with CUX2 (though based on DARTFISH and FPKM it should be) [[File:20150720_RNAFISH2_488.jpg]] *Sample 2 - 647 (CUX2) **Image is very grainy making it hard to tell distinct spots from noise, probably needs better excitation parameters [[File:20150720_RNAFISH2_647.jpg]] ====Zeiss Microscope in Huang Lab with Metal-Halide light==== *7/22/2015
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