Daniel:Notebook/RNAFACS/2015-10-21: Difference between revisions
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So long story short, we'll give it another go with lower amount of pretreat. | So long story short, we'll give it another go with lower amount of pretreat. | ||
'''Samples''': From Andrew: Cerebellum, May 2014, Patient | '''Samples''': From Andrew: Cerebellum Contol, May 20 2014, Patient 7-03 3340/uL ~5 uL | ||
==Nuclei Adhesion== | ==Nuclei Adhesion== | ||
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##May want to retrace hydrophobic barrier; can skip | ##May want to retrace hydrophobic barrier; can skip | ||
#Apply Pretreat 3 | #Apply Pretreat 3 | ||
##'''Dilute pretreat 3 1:15; 1 drop of pretreat 3: ~ | ##'''Dilute pretreat 3 1:15; 1 drop of pretreat 3: ~25 uL; add 700 uL 1X PBS to 2drops pretreat 3''' | ||
##Remove excess liquid; add '''150 uL drops of diluted pretreat 3''' | ##Remove excess liquid; add '''150 uL drops of diluted pretreat 3''' | ||
##Incubate for 10 min at RT (in oven) | ##Incubate for 10 min at RT (in oven) | ||
Line 110: | Line 110: | ||
#Store in the dark at 4C | #Store in the dark at 4C | ||
#Recommended Viewing at 20-40X magnification | #Recommended Viewing at 20-40X magnification | ||
==Microscopy Results== | |||
Yeah, nothing was on the slides. As in, I found no nuclei, let alone any signal. I think Andrew's samples are too degraded (they are over a year old) to be able to do this analysis. However, Blue gave me some samples today, and I will try this test again [[Daniel:Notebook/RNAFACS/2015-10-22|tomorrow]] with those samples. | |||
[[Category:RNAScope]] [[Category:RNAFACS]] [[Category:20151021]] | [[Category:RNAScope]] [[Category:RNAFACS]] [[Category:20151021]] |
Latest revision as of 15:23, 22 October 2015
RNA Scope Trial Run 4: Pretreat[edit]
So after talking with acdbio people yesterday, they recommended that I reduce the stringency of my pretreat. I did have success over the weekend with the pretreat as usual, but there were only a few nuclei to look at, which could be the result of several things:
- Over-pretreating
- Bad adhesion (shouldn't be a problem, since other times had loads of nuclei)
- Low nuclei count in Blue's sort samples (possible, but not much I can do on that one)
So long story short, we'll give it another go with lower amount of pretreat.
Samples: From Andrew: Cerebellum Contol, May 20 2014, Patient 7-03 3340/uL ~5 uL
Nuclei Adhesion[edit]
- Clean glass slide in 100% EtOH; allow to air dry
- Draw a square around the desired adhesion area using a hydrophobic barrier pen
- Remove cells from -80C
- Thaw, and add 500uL of 4% paraformaldehyde to the tube and wait 2 min
- Centrifuge cells for 5 min at 1000g
- Remove supernatant, careful not to disturb the pellet
- Add 500 uL of 70% EtOH
- Centrifuge cells for 5 min at 1000g
- Remove supernatant, careful not to disturb the pellet
- Resuspend nuclei in 20 uL 70% EtOH
- Deposit on to glass slide
- Use a coverslip to gently spread the 20 uL across the slide
- Be careful not to scratch the slide
- Dry the slide using a heating block at 60C for 10-15 min
RNA Scope[edit]
Sample Matrix
Slide 1 | Slide 2 | |
C1 | RBFOX3/NeuN | +Control |
C2 | -Control | +Control |
C3 | FEZF2 | +Control |
Nuclei Prep[edit]
- Rehydration
- Incubate slides in 70% EtOH for 2 minutes
- Incubate slides in 50% EtOH for 2 minutes
- Incubate slides in 1X PBS for 10 minutes
- May want to retrace hydrophobic barrier; can skip
- Apply Pretreat 3
- Dilute pretreat 3 1:15; 1 drop of pretreat 3: ~25 uL; add 700 uL 1X PBS to 2drops pretreat 3
- Remove excess liquid; add 150 uL drops of diluted pretreat 3
- Incubate for 10 min at RT (in oven)
- Flick to remove excess liquid
- Rinse in fresh 1X PBS; be sure to agitate to clean well
Fluorescent Assay[edit]
- Prepare-Reagents
- Prepare probes by warming to 40C for 10 minutes, then cool to RT
- Briefly spin down C2 probes to collect the liquid
- Combine C2 and C1 probes by pipetting into a new 1.5mL tube at 1:50 ratio
- Prepare 1X Wash Buffer
- Place AMP1-4 FL reagents at RT
- Ensure HybEZ OVEN and Control Tray are at 40C
- Hybridize Probe
- Flick to remove excess liquid; add 2-4 drops of probe
- Incubate for 2 hrs at 40C
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- AMP1-FL
- Flick to remove excess liquid
- Add 2-4 drops of AMP1-FL; Incubate for 30 minutes at 40C
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- AMP2-FL
- Flick to remove excess liquid
- Add 2-4 drops of AMP2-FL; Incubate for 15 minutes at 40C
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- AMP3-FL
- Flick to remove excess liquid
- Add 2-4 drops of AMP3-FL; Incubate for 30 minutes at 40C
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- AMP4-FL
- Flick to remove excess liquid
- Add 2-4 drops of AMP4-FL ALT C; Incubate for 15 minutes at 40C
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- Counterstain and Mount
- Flick to remove excess liquid;
- Add 2-4 drops DAPI; Incubate 30 sec at RT
- Remove DAPI and immediate place 1-2 drops of fluorescent mounting medium
- Seal with a coverslip; avoid bubbles
- Store in the dark at 4C
- Recommended Viewing at 20-40X magnification
Microscopy Results[edit]
Yeah, nothing was on the slides. As in, I found no nuclei, let alone any signal. I think Andrew's samples are too degraded (they are over a year old) to be able to do this analysis. However, Blue gave me some samples today, and I will try this test again tomorrow with those samples.