I am currently extracting some blood samples and would like to know detailed protocol and some hints.
I usually isolate RNA by using QIAGEN - RNeasy Mini Kit from fresh blood ot frozen tissue samples.
I think that never have been isolate RNA from frozen blood but it could work. But, the best would be to mix the blood with PAX solution if you want to store for long time, and then use PAXgene Blood RNA Kit to extract RNA
Hi Sardar,
You have two options here, as for most of the cases, an RNA extraction kit, or the clasic Trizol, TriZ method. Both methods are good and reliable, but they differ mainly in the amount of work and volume of sample required. The Trizol extraction method takes a bit longer, but it is cheaper. In the case you have not much sample, i would trend to go for a Kit, ideally a mini prep kit, as suggested by Mario. Regarding your frozen samples, if they have been stores at -80 C it should not be an issue at all, and both methods should gave great results!!.
Cheers
For extract RNA from fresh blood you can use a Trizole or RNAse Mini Kit by Qiagen, while for extract RNA from frozen blood you can try with Phenol-guanidien method.
This is how I extract RNA from blood...
1)Layer Blood carefully over an equal volume of Histopaque(Sigma) in a centrifuge tube(I use a 15ml tube)
2)Centrifuge for 20 min at RT
3) aspirate the PBMC layer(formed as a white ring below the plasma layer)
4) Wash the PBMC layer with 1X PBS twice or thrice.
5)Add 1ml TriZol and mix well to dissolve th pellet and keep it for 3 min
6)Add 200ul chloroform and mix well. and centrifuge
7) Transfer the aqueous layer to another tube and add equal volume of isopropanol
8) Incubate at -20 for 20 min
9) centrifue and discard the supernatent carefully
10) wash pellet with 70% ethanol for twice and airdry the pellet
11) Dissolve the pellet in 30-50 ul Nuclease free water.
Dear Sardar,
I think these papers can help you:
https://www.ncbi.nlm.nih.gov/pubmed/27597528
https://www.ncbi.nlm.nih.gov/pubmed/24567882
Good luck, Hamid
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