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.
ps: and anybody know how I can improve reproducibility of Trizol extraction?
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:(