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RNA extraction from seawater filters-welcome to the challenge - (Feb/19/2008 )

All right, this is kinda desperate and I could use all advice:

I am trying to isolate RNA from a membrane filter with 10L seawater filtered on it and the filter contains appr 10^7 cells. I am doing ACGP extraction, so what I tried so far is to get the whole filter in a falcon tube and immerse it in 10ml Trizol -self made, works perfect for everything else including cells on smaller filters-, vortex till filter looks clean and aliquoting the solution in eppis to carry out with chloroform extraction... I see that I have ~300ng per ul RNA with nanodrop per aliquot, but on the gel I saw nothing so I decided I have nothing since the cell number is so low. But this situation is not getting better so I have to find a way to get the small amount of RNA from the large filter (15cm) which requires that I have a lot of volume for my lysis solution. I cannot filter more water on one filter cause mRNA degrades in the meanwhile. I cannot carry out the extraction in the 50ml falcon tube because then the centrifugation speed is not enough. I cannot pool the aliquoted RNA and precipitate because I lose material in the meanwhile.

Any suggestions?
ps: and anybody know how I can improve reproducibility of Trizol extraction?


-emiliania huxleyi-

Could you recover and culture the cells first then extract the RNA?


no, the idea is exactly to work culture-independently because only %1 of the bacteria in seawater are culturable:(

QUOTE (chessplayer @ Feb 20 2008, 05:58 PM)
Could you recover and culture the cells first then extract the RNA?

-emiliania huxleyi-