Notes, Thoughts, and Ideas on RNA isolation (and thoughts from Sam)
Spoke with Sam the other day about everything we’ve tried in regards to extracting RNA from our crab hemolymph samples. He shared a lot of ideas of potential next steps for figuring out ….
Google drawing with history and info on all RNA extractions thus far
Trizol LS is a concentrated form of Trizol. It is concentrated so that it can work with liquid samples. If regular Trizol is used in liquid samples, the salts in the Trizol become overly diluted, which is not good because the salts are important for lysis and separation of DNA and RNA.
I apparently used geoduck supernatant over the weekend in my RNA extraction attempts. I used the samples “1H” and “2H” that Shelly said I could use from her box. I took out 250ul of sample from those tubes to use in my extractions. Apparently that was the supernatant from the spinning down of the hemolymph. The pelleted cells are in tubes “1H 11-08” and “2H 11-08” and are what I should have used.
Ideas on potential next steps from Sam:
Direct comparison between crab hemolymph and geoduck hemolymph
Have someone who is already going to Pt. Whitney take some hemolymph samples from geoduck. Same volume of hemolymph as the crabs was taken. Preserve in same amount of RNAlater. Put on dry ice/liquid nitrogen. Bring back to UW and I will processs in same manner as I have processed the crab hemolymph - spin down to pellet, use one or several of the extraction protocols to attempt to extract RNA
Get a live crab to keep in lab and try pulling out hemolymph and use the fresh hemolymph
Try Direct-zol RNA MiniPrep with pelleted crab hemolymph, corresponding crab supernatant, and geoduck hemocytes using Trizol LS
Use 2 crab hemolymph pellets, 2 corresponding crab superanatant, 2 geoduck hemocyte pellets
Add 100ul of DI water to pelleted crab and pelleted geoduck hemolymph
Prepare samples using Trizol LS. Resuspend in 25ul of RNase-Free Water
For the supernatant samples
Supernatant preparation will need to happen in a 15ml conical tube (much more sample to use). Will also need to run the sample through the spin column multiple times
Vortex then separate into multiple tubes to add chloroform, mix, spin down.
Then resuspend RNA in 20ul of RNase-free water, suck it up in the pipet, go to the next tube, pipet to mix, go to the next tube pipet to mix… so that all RNA is in one tube in 20ul.