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Direct RT-PCR from Frozen Cells?


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#1 jah

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Posted 16 April 2009 - 05:06 AM

I was wondering if anyone had ever tried to extract RNA from cryopreserved tumor cells (DMSO-based media)....without resuscitating them first. The reason I as Is that I just need a quick qualitative (yes/no) answer about whether one of our banked cell lines expressed a particular transcript.

When the cells were banked, they were about 70% confluent and actively proliferating, so about as healthy as can be when frozen. Rather than resuscutate the cells and wait several days for equilibration, I thought I could just thaw the frozen cells on ice, spin to pellet the cells, and then perform the lysis and extraction directly. Any thoughts?

#2 gfischer

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Posted 16 April 2009 - 07:02 AM

I was wondering if anyone had ever tried to extract RNA from cryopreserved tumor cells (DMSO-based media)....without resuscitating them first. The reason I as Is that I just need a quick qualitative (yes/no) answer about whether one of our banked cell lines expressed a particular transcript.

When the cells were banked, they were about 70% confluent and actively proliferating, so about as healthy as can be when frozen. Rather than resuscutate the cells and wait several days for equilibration, I thought I could just thaw the frozen cells on ice, spin to pellet the cells, and then perform the lysis and extraction directly. Any thoughts?


This should work fine. I've done it before, and had success.
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