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RNA extraction from frozen cells suspended in PBS - (Nov/21/2008 )

I'm trying to extract RNA from human lymphoblasts. I made the mistake of freezing my cells suspended in of PBS (about 3-4million cells in 300ul of PBS - frozen at -80). I'm afraid that the cells will lyse due to freezing and thawing, causing the RNA to degrade during thawing because of a lack of RNase-inhibitors and causing the cell lysate to be diluted in PBS making the lysis buffer less effective at lysing the cells that survive the freeze/thaw.

As far I as I can tell, I have two choices on how to proceed:

1) add lysis buffer to thawed PBS-suspended cells and risk a diluted buffer
2) Spin PBS-suspended cells down to remove PBS after thawing and risk losing RNA from cells lysed by freeze/thaw

Any ideas on which approach would be better, or if some other approach would be best to get a decent yield of RNA?

-joe_chi-

QUOTE (joe_chi @ Nov 21 2008, 06:29 PM)
I'm trying to extract RNA from human lymphoblasts. I made the mistake of freezing my cells suspended in of PBS (about 3-4million cells in 300ul of PBS - frozen at -80). I'm afraid that the cells will lyse due to freezing and thawing, causing the RNA to degrade during thawing because of a lack of RNase-inhibitors and causing the cell lysate to be diluted in PBS making the lysis buffer less effective at lysing the cells that survive the freeze/thaw.

As far I as I can tell, I have two choices on how to proceed:

1) add lysis buffer to thawed PBS-suspended cells and risk a diluted buffer
2) Spin PBS-suspended cells down to remove PBS after thawing and risk losing RNA from cells lysed by freeze/thaw

Any ideas on which approach would be better, or if some other approach would be best to get a decent yield of RNA?


Split the 300┬Ál and try both ways in parallel... :-)
....it's the savest choice....should be enoungh RNA for cDNA-Synthesis and qPCR

-K_R-

Yes. split to be safe. If you don't have a choice, extract the quickest way possible: spin down and go. No matter how once the RNA is outside the cell it's not much use in your study....

-chrisbelle-