I have some patients cryopreserved lymphocytes samples which I want to analyze with RT-PCR later. It has been stored in the liquid nitrogen tank more than 2 years. I also want to use them for FISH, so I thawed them first, then aliquoted them into two tubes; one is for FISH, the other is for RNA extraction.
Then I tried RNA extraction of some of the samples, but I could not get RNA, almost at all.
The questions are
"Is it difficult to get the RNA out of these samples?"
"Can I stimulate and culture these lymphocytes for a while and get the RNA out of them?"
" What is the best way to isolate RNA from the small amount of samples?"
The samples have been stored in 90%FBS, 10%DMSO.
Thank you for your help in advance.
Dear Yuka
1-It is not difficult to extract RNA from your samples at all!
2-You may be able to culture these cell (difficult to say) but remember that the RNA profile of cultured cells are different from natural tissue.
3- RNeasy Micro Kit from Qiagen is very good for small amount of tissue or cells.
Good luck
Hi Ali,
Thank you for your answer.
I used the QIAGEN RNeasy mini plus kit and could not get anything out of it.
Do you think it is because I thawed the sample for aliquoting so that RNase digested almost all the RNAs?
My samples are 8 years old.
I will check RNeasy micro kit!
Thank you,
Yuka
Before you thaw cells, add some RNALater in the frozen vial, thaw it, spin it, add 200ul RNALAter to the pellet and than spin it and proceed the pellet with your RNAeasy kit. RNALater will protect any RNA degradation if it might be occurring
Hi Subhash,
I will try that way once I get RNALater reagent, and get back to you.
Thank you
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