Iv been having a lot of trouble recently losing RNA during my Trizol RNA Extractions and subsequent precipitations (with Sodium Acetate and Ethanol).
In every group, there's always a few samples where the yield is a fraction of the rest of the samples.
I know it's not the cells themselves or lysis. So most likely its during the aspirations.
The biggest issue is that sometimes ill see a white pellet and other times i wont see anything. Sometimes as in, between trials, but im not changing anything in my protocol so its mind boggling. And just in general aspirating/sucking out the 70% ethanol, I must be sucking out RNA.
I go from the opposite side of the tube as to where the RNA should be. I tilt the tube first to suck out most of it before i bring the pipetor/aspirator closer to the bottom to get the rest of the ethanol out, but since i cant see the pellet, its hard to get the rest of the ethanol out without risking sucking out the pellet.
Does anyone know what determines whether you see a white pellet or not? I know if you overdry itll turn transparent/translucent, but im talking when the RNA is still in 70% ethanol.
I keep losing samples and its driving me nuts.
Do you mix well after adding the sodium acetate and ethanol? That step can determine your efficiency of RNA precipitation.
Instead of aspirating the the ethanol/solutions, just try to toss/invert the tube to remove solutions. sometime the pellet are very loosely attached to the tube, even with careful aspiration they tend to come out.
I've done this with stem cells a bunch of times. The main problem is probably the amount of cells (with Trizol you want 2x10^6 or more cells per RNA extraction). When I perfrom this with 5x10^5 cells I often can't see a pellet. You can add 1ul glycogen if seeing a pellet is important for your aspirating technique (I use a p1000 pipettor and then a 20ul pipettor to get teh rest of the ethanol after the precipitation and wash).
Don't take everything after the precipitation step, leave about 20-50ul 100% EtoH and then add a ml of 70% EtOH. This will reduce the chance of sucking up your RNA. Also spin at full speed in your centrifuge, not the 12000xg they recommend. This will pellet your RNA better if you are not seeing a pellet and reduce the chance that it'll come off the tube.
How much RNA per cell are you getting on a good run (should tell you how many cells you will need to get a 'good' amount of RNA)? When you isolate your RNA do you see a good 260:280; 260:230 O.D.? Do you see peaks at 280 and 230 (if so this is Trizol contamination; do a chloroform: isoamyl alcohol cleanup followed by sodium acetate precipitation.
Spinning long and hard is the best thing you can do, I think, to increase yield (see attached). I would add glycogen for easy visualization and better precipitation and use a pipette for gentle aspiration.
Nice stuff, Brian.
RE cooling everything to -80C, see my previously attached if you're interested. The thinking generally is that colder solutions are less effective solvents therefore precipitate out more RNA, however this paper argues that the increased viscosity makes extreme cooling a net negative on efficiency.
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