RNA extraction from FROZEN blood - (Dec/19/2007 )

Hallo,
I need to extract RNA from frozen blood (immediately frozen in dry ice and then stored at -80°C). I know Qiagen PaxGene kit and RNeasy Blood kit but boths have limitations in my case: 1) PaxGene kit is quite expensive and I need to take 200 ovine blood samples for several times (5 times I suppose, about 1000 blood samples), therefore a huge number of samples and moreover not all samples will be processed (it depends whether the animal will get the disease); 2) RNeasy Blood kit must be used with fresh blood.
I would like to know whether someone know a protocol to conveniently extract total RNA from frozen whole blood.
Thank you for your help !

Simone

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Can't you just use Trizol?

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Yes, it could be a solution. I have not yet started the work and I was looking for someone with experience in extracting RNA from frozen blood. Someone has told me that RNA degrades even if blood is immediately frozen in dry ice and stored at -80°C. Cen you help me ? Thanks !!!

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I haven't extracted RNA from frozen blood but I have from organs and bone marrow. I froze the tissue/BM in liquid nitrogen (to preserve RNA) and then proceeded with the trizol protocol. It always worked. If I were to do it from frozen blood I would suggest keeping your blood frozen until you're ready to go, then thaw it on ice before adding trizol. Alternatively - are you looking to isolate RNA from any particular cell type?
Have a great festive season
Clare

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I need to extract RNA from the whole blood and not from a single cell type. Can you send me your trizol protocol please ? Someone else can help me ? I read on the forum that a user named Auriolw was trying different protocols on the same matrix (ovine frozen blood) with little results. Does someone know him/her ? I would like to get a contact, the link to that past discussion is pastes below:

http://www.protocol-online.org/biology-for...posts/9938.html

Thanks !!!

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go to your messaging page and send a pm to the member.

you can get to the messaging page by clicking on the upper right "new messages".

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Just use the protocol that comes with the bottle of Trizol

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RNA content in whole blood is very low, you might be able to get 1-2 ug/ml blood for most methods or kits, including trizol. Without removing hemoglobin, you would likely get little RNA. It seems that RNA binds to hemoglobin and gets lost with it. If you have thousands of samples, I'd recommend the scalable aqueous solution based AquaRNA and its RBC lysis solution, which works for both fresh and frozen blood.

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