A lot of protocols say to avoid washing cells with PBS before addition of TRIzol as this increases the chance of RNA degradation.
An example is the official TRIzol documentation:
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/trizol_reagent.pdf
"Note: Do not wash cells before addition of TRIzol™ Reagent to avoid mRNA degradation"
And I see the same thing on a lot of other TRIzol extraction protocols.
However, a coworker told me that I must wash my cells to remove the dead cells. And if I don't wash, I'll be getting RNA from those dead cells (in addition to the live cells), which could obscure results in some experiments.
Is that true?
I don't want inaccurate results or RNA degradation...
There will be NO problem in washing cells with PBS before adding TRIzol. We do also wash sometimes & sometimes not. Even you can try to remove spent media from the pellet as much as possible and go ahead without washing.
I recommend to think about of quench the cell metabolism. If you work with a early stimulation time, the time spent to wash cell can interfere in final result and increase the difference between biolofical replicates.
Like Saurabh mentioned even we have not encountered any problem by using both the methods. As a matter of fact many times we have directly lysed the pellet in TRIzol after we have pelleted the cells down. Other times we would just do one wash with 1X PBS and upon removal add TRIzol. Just one observation is that the RNA yield has been better when washing with PBS was not done!!
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