Matt:LabNotes/2015-6-18

From ZhangLabWiki
Revision as of 02:55, 26 June 2015 by >Mzcai (→‎Results)
Jump to navigation Jump to search

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)

RNA FISH Procedure

  • Sample 1 labeled: "RNAFISH1 6.18.2015"
    • 488 and 594
  • Sample 2 labeled: "RNAFISH2 6.18.2015"
    • 546 and 647
  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 (2X volume)
    • Hybridization Buffer 200ul
    • Probe 10ul of each (5ul-488+5ul-594 and 5ul-546+5ul-647)
  4. Wash the cells with 1ml of PBS (RNase free) 2 times
  5. Add 1ml Wash Buffer and let sit 10min at RT
  6. Aspirate
  7. Add 110ul hybridization solution to each sample and incubate overnight at 37C (~18hrs)
  8. Warm up 6ml Wash Buffer and 2ml Wash Buffer with DAPI to 37C
  9. Rinse with 2ml Wash Buffer
  10. Add 1ml Wash Buffer and incubate 30min at 37C
  11. Replace with 1ml Wash Buffer with DAPI and incubate 30min at 37C
  12. Wash with 2X SSC (warmed to 30C) twice
  13. 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"

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)