Hi Everyone,
I have a question about RNA isolation using Trizol with cells that are in suspension after cell count. Whenever I extract RNA from cell culture I always add the Trizol directly to the monolayer of cells, scrape, and transfer the lysed cells into a fresh eppendorf tube. My question is, I have been asked to get a cell count for the samples as well. I could accomplish this my either having a 6-well plate set up for only cell count (seeded the same density as the experimental conditions) or I can trypsinize the cells, spin down with centrifugation, and resuspend in medium to conduct a cell count. Than respin down into a pellet, remove supernatant, and add trizol directly to the pellet. Anyone done this before without losing mRNA quality, or should I just go with my traditional method of adding Trizol directly to the monolayer?
Thanks
Both adding Trizol directly to cells in a 6-well dish, and to a washed pellet of cells is equally acceptable. I don't believe you will observe any loss of quality, perhaps a bit higher quality in the washed method, since you are removing metabolites and debris.
Good luck!
Dear Shawan,
You can isolate RNA by trizol method from pelleted cells after trypsanisation without comprimising in the quality. Ihave done both from monolayer direlctly and after trypsanisation never had a issue withe the quality of RNA isolated.
Hi, Shawn Krueger
As other already told you, no problems with both ways. Just an extra piece of advice, samples manipulated are always prone to problems (or some bias). So, what are you intending to detect/quantify? If it is associated with morphological mRNAs, maybe the time and treatment may change your expression profile. So, my advice is to evaluate it in a small sample before you decide which method is better for your application. Ok?
You could get three samples and do it in parallel and check your expression profile of interest.
Hope it helps you.
All the best.
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