Hello
I’m about to extract total RNA from cell line using TRIZOL ... is it safe to scrab first cell and put the trizol then freezing and complet the rest of proceduer next day ..
or scrab cell and leave it will the PBS and freezing and next day centrifuge to remove PBS and start trizol procedure !!!??
which cases are safe for my work ??
Greetings
thanks in advance
Zaynab
Leaving cells in TRIzol and freezing them was what we did before, and it worked well. But I think if you only leave the cells overnight, both of them should be fine.
If you have the option of leaving cells frozen, or frozen in trizol, use the latter: they'll still be frozen either way, but with the addition of trizol, they'll also be thoroughly lysed, denatured and quenched of residual RNAse activity.
Freeze them at -80, though (NOT -20).
I often scrape cells directly into trizol (add trizol to well, scrape, collect resultant suspension), and have successfully isolated qPCR-quality RNA after a year of storage at -80.
If your are going to isolate the RNA for your RT-PCR study, i will recommend you to add trizol reagent into the scraped cell and stored at -80c. Next day you can thraw the cell and can start with your remaining process.
However, if you place cell in 1X PBS and stored in -80c. Next day when you will do thrawing of cell, due to rupturing of cell membrane of cells all protein will come in the PBS. Then no use of adding Trizol reagent.
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