extract RNA from frozen cells - (Jun/30/2005 )
hi
I am extracting RNA from hela cells at the moment. I was wondering whether i can extract RNA from frozen cells rather than fresh cells. Anyone have done this before? will this affect the RNA quality?
I am extracting RNA from hela cells at the moment. I was wondering whether i can extract RNA from frozen cells rather than fresh cells. Anyone have done this before? will this affect the RNA quality?
Hi
RNA is very sensitive to freeze thawning cycles. We've experience that about 25% degradates per cycle. But it is a good way to disrupt your cells.
If I had choice, I would do from fresh cells. If not, it should work anyway, but you might have a little bit less in your tube...
Hope it helps..
We extract RNA from frozen tissue samples all the time and get good results... don't know why it would make a difference if it were cells.
Having said that I would also agree that collecting RNA from fresh cells would be best.
hi
i use trizol on frozen cells (-80° required) and heat it at 70° to put it heated on frozen cells (just after they've been put out from the -80°)
Hi guys, thank you very much for your advice. To fred 33, why u heat the trizol before use it on frozen cells? also when i run my RNA on the gel, i observed a third band which is much smaller than the two bands i should get. so it can't be the genomic DNA. this band is not as strong as the other two bands. Does this mean my RNA is degraded? will this affect my RT-PCR
Hi,
The lower band is probably the 5S RNA band. This is very common.
Also, it is indeed fine to freeze cells. But, I would recommend freezing the cells
in your lysis buffer.
In fact, we have found that freezing cells in lysis buffer (Guanidine Isothiocyanate (spelling??)) increases the amount of total RNA we recover. So we always freeze our cells. Increased recovery is most likely due to disruption of intracellular membranes which actually contain a high amount of mRNAs. But, if you are using a kit with Hela cells you get tons of RNA anyways, so it doesn't matter what you do so long as you don't spit in the tube 
.
There have also been some discussions about whether freeezing in lysis buffer actually increases stability but I am not convinced of this at all. BUT, I have witnessed RNA purposely left at 65 degrees for 4 hours just to prove how stable it was, and it gave a perfect gel. But you probably don't wanna try this.
hi
i put trizol pre heated on cells to enhance defrost of the cells and lysis. Cells do not get time to thaw and begin degradation of DNA/RNA and others... in brief, reduces degradation.
cheers.
fred