I need to extract mRNA from my cells but I am not sure if Trizol, a corrosive product, is compatible with the culture 12-well plates. May I should trypsinize and transfer the cells to a eppendorf before add the Trizol?
I usually do one (RNAse-free) PBS wash, then apply the Trizol reagent directly to the culture plate. The cells either come off immediately and I aspirate the Trizol into microbtubes, or for more adherent cells I might have to swirl a little or give a scrape and they all come off, then I put the cells/Trizol suspension into the microtubes. Trizol seems to work fine when applied to culture plates, and I have never had issues with downstream applications.
Hi Elisabet. Yes, it usually does the work with 12-well plates. I extracted mRNA from HEK293T cells and HeLa cells grown in 12-well plates. I could even see the white mRNA precipitates in the tubes after extraction. So I think the extraction process is mainly dependent on cell lines not on the plates. Some cell lines grow well and can expand the whole well (more than 90 confluence), and you can get enough mRNA for your later experiments, some cells may not. For some cells, if they could not adhere the plate well, you can add the Trizol regent directly to the well after you aspirate the media. And you can use the Trizol regent to lyse the cells with the pipette in the wells. Thanks.
Thank you Carmen and Shuaipeng. I will do it as you say. :)
Elisabeth.
Bellow is my way how to process your cells for total RNA isolation.
1. Have a bucket ready with ice on the bench top.
2. Take dishes with cell culture out of the incubator.
3. Place them on the ice.
4. Gently aspirate the medium.
5. Add cold PBS (4oC) and gently rinse dish(s).
6. Gently aspirate PBS.
7. Immediately add cold (4oC) Trizol reagent (volume depends on the size of dishes ; I use 1 ml per 100mm dish, for example). Make sure to cover entire dish/cells area.
8. Wait ~1min (dishes are on ice).
9. Harvest cells using Pasteur glass pipette. Cells and Trizol will be kind of gluey at this point. For larger dishes (35-100mm) use scraper instead of Pasteur pipette to collect the cells. (Eppendorf) pipet with 1ml tip will work, too.
10. Transfer cells into 1.7-2.0ml Eppendorf tube and process them for further steps.
This is my way how to work with Trizol reagent. And is working all the time.
More datails at https://tools.thermofisher.com/content/sfs/manuals/trizol_reagent.pdf
Good luck
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