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Combining Paxgene preservation with alternate RNA extraction method

RNA isolation trizol paxgene

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#1 lkom

lkom

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Posted 09 February 2014 - 10:06 PM

Hi,

 

I have whole blood samples that were preserved via Paxgene tubes, but I am using an alternate method for RNA extraction because the downstream Paxgene kit only produces degraded RNA (due to nucleated RBCs and associated high amounts of proteins, etc.). I have had success using trizol extraction on the pellet, followed by column based clean-up to improve purity, but I want to improve my yield and quality.

 

To do this I'm trying to figure out what's happening when the sample is in the buffer but I am finding conflicting info. Does the Paxgene buffer actually lyse the cells (that's what their manual suggests), or is that at a later step (since you spin down the pellet of cells presumably that get transferred to next step and then lysed)? Some have people have said they were successful adding Trizol LS or Tri Reagent directly to 500ul of the sample (blood+paxgene buffer) and following that protocol, but others have said this won't work because the paxgene buffer will inihibit cell lysis by Trizol LS, etc.

 

Any insight or suggestions are much appreciated!







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