total RNA isolation question - (Jul/24/2006 )
I use Tri-reagents to do total RNA isolation. If some cells are dead, can their total RNAs be isolated also?
Thanks in advance!
I guess RNA from cells dead before extraction would be rapidly degraded, but I'm not sure.
If the cells are still reasonably intact and the RNA has not begun degrading then you can probably isolate RNA.
The real question you should be asking (and I would if I was a referee or examiner) is does the RNA population in your dead cells represent the RNA population in the healthy cells. The answer is probably no. Therefore your total RNA will be a mixture of two separate RNA populations. If you want to perform RT-PCR or qPCR you cannot be sure that your results are representative.
You need to minimise the amount of dead cells included in your prep (unless it nearly 100% - then you need to minimise the living ones)
wash twice with PBS your plated cells in order to remove almost all dead ones.
I thought you were not supposed to wash the plated cells before the addition of Trizol, as that can significantly reduce RNA quality?
well what i do is wash cells twice, trypsinise, pellet, wash twice, measure roughly volume of the pellet and add 10volumes of trizol. works fine
Well, Fred, now I can't find where I read that - but good to know that it is ok to wash!
I agree with him.
i would add that my PBS is at 4° for these washing steps... and the centrifuge too