total rna extraction from whole blood - for rt pcr (Jul/27/2004 )

hi!
i am working on RT PCR for PPR VIRUS. for this i need to extract total rna from whole blood and nasal swabs . have any of you tried TRI Reagent (sigma) for total rna extraction from whole blood ........i tried it with 200 microliters of blood ........but no rna.......
if any of you have experience with this ...plz reply me.
ashish

Hi

I use Tri reagent for neutrophils that have been isolated from whole blood. I normally have 10 x 10^6 cells / RNA extraction. You may not have enough cells in 200ul cause a lot of that will be serum etc. The data sheet with the Tri might tell you how much whole blood you need. Otherwise your RNA may be degrading. I find that any red blood cell contamination in my neutrophils causes the RNA to degrade.
I quantify my RNA on a spec and then run it on a gel to check its integrity.

Hope this helps

try using double amt of starting material, i do it that way
Other way is to use RNeasy kit by Qiagen for total RNA extraction (use 350uL ) of starting material in this at least
preeti

Hi, i use Trizol (Invitrogen) for bacterial RNA isolation, i think they do a Trizol for tissues (LS?). Basically i have found that with Trizol the secret is to shake vigourously, for this i resuspend my cells in Trizol and bead beat them in it. Hope it helps. Also i highly recommend using RNase Away Sprays, DEPC treated water and store your RNA in DEPC treated water if you wish to use it for PCR, i used a buffer for storage which contained a chelating agent and my PCR's failed because the MgCl has been chelated!