RNA extraction from sludge - (Nov/10/2005 )

Hi everybody !

I'm trying to extract RNA from sludge samples coming from different wastewater treatment plants and I have lot of troubles. I'm using a classical protocol with a mechanical lysis with beads and a beadbeater and hot phenol and then I do several phenol/chloroform extraction (up to 5 in some cases) and finally 2 chloroform extraction to remove phenol. Then I precipitate with ethanol at -80°C. After precipitation, I wash the pellet with ethanol 80 % and dry it before dissolution in DEPC-treated water.

The problem is I never get the same results from one experiment to another... Sometimes I have a final ratio 260/280 of 1.7 but sometimes it is more about 1.3 and in that case I can't see anything on agarose gel.

I also have a lot of DNA hard to digest (about 10 µL of DNase I required for a 50 µL sample !!). Do you have any solution to limit the presence of DNA ?

I tried to lower the pH to 4 (instead of 5 initially) with no effect on the DNA quantity. I don't know really the effect of pH cause in several RNA extraction kit I noticed that the pH of buffers are at 8 even for RNA extraction. Does it matter really ?

Second problem : my samples have been frozen before any manipulation. So I have for each sample 50 mL of frozen sludge. To treat these samples i need to thaw them and then centrifuge, wash, resuspend, aliquot in smaller tubes and then extract... The problem is that I think the thawing phase will degrade the major party of my RNA. Do you have any suggestion to prevent the degradation during thawing ? Maybe phenol could protect the RNA from RNase ?

Thanks a lot for your answers

Hi friend,
what organism you are looking for, bactteria or viruses?
Your RNA purity is low, how about the RNA concentration?

Best regards

I'm looking for bacteria to do dot-blot experiment.