Daniel:Notebook/RNAFACS/2015-9-26
RNA FACS Using RNA Scope
We are pivoting the protocol for RNA FACS based on information Dr. Zhang got over the summer that the smFISH protocol has a major flaw: it has weak signal strength. Only genes with very high expression can be visualized, and even then it is tricky. Since the FACS protocol calls for strong signal, this won't work. RNA Scope's major advantage is in it's amplification of weak signals, making each probe considerably brighter and requiring fewer binding events to detect a signal. So, we'll be trying that.
Nuclei Adhesion
First thing is first, I want to make sure that my protocol for nuclear adhesion is a valid one. Since I won't be using any probe for this, I'll stick to the mouse nuclei. Once I am sure of the efficacy, I can try once with human nuclei to make sure they work as well. Most papers I've seen require fixed nuclei, and since I am not using fixed nuclei, we'll start with that.
- Clean glass slide in 100% EtOH; allow to air dry
- Remove cells from -80C and spin down for 5 min at 1000g
- Remove supernatant, careful not to disturb the pellet
- Add 500uL of 4% paraformaldehyde to the tube and wait 5 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
- Add 15 uL Slowfade + DAPI and seal with a coverslip
NOTE: I'm not actually doing the protocol today. I need to secure access to the Huang lab microscope (although I can do this check using the confocal) and I want to get the protocol down for RNA Scope before I do the experiment. Probably Monday.