Hello all,

I am doing total RNA extractions on bacteria (E. coli BL21*) and I am stunned by the amount of reagent necessary for the extraction.

According to the Invitrogen protocol for their Trizol Reagent (Cat. No. 15596-018), 1 mL of Trizol is necessary for 5-10x10^6 cells either these cells are plant, animal or bacterial cells.

Using bacteria, It meens that I have to use 5 mL of Trizol for a tiny (~20-50 µL) pellet!!! (This pellet is obtained by hervesting 1-2 mL of 0.5-1 OD600 culture.

Moreover, bacteria cells are ~1000 times smaller than plant or animal cells, so I would believe that one could use 1000 times (or at least 100 times) more bacteria cells than plant or animal cells. But I have asked the technical support and of course they told me to keep the indicated (very consumming) volumes...

So, is anybody has ever tried to reduce the amount of Trizol and checked that everything worked fine?
Or, id anybody has an idea of why we cannot think in terms of mass amount but rather in terms of number of cells?

Thanks a lot for your help!

Alex

Invitrogen should hire you to rewrite their manual. Use your common sense and ignore their absurd advice.

AquaRNA (http://www.aquaplasmid.com/AquaRNA.html) is really the best and simplest reagent for bacterial RNA extraction. Email info (at) aquaplasmid.com for a free sample to assure you self.

1) Pellet 0.5ml bacterial culture
2) Incubate in 100ul lysozyme/TE for 15-30min
3) Add 100ul AquaRNA and incubate on ice for 5-15min
4) Add 200ul isopropanol and spin 5min to pellet DNA/RNA
5) Rinse pellet with 75% ethanol
6) Add 100ul diH2O and spin 5min to pellet insoluble
7) Transfer clear DNA/RNA solution to a new tube

You cannot find anything that is as simple as this one. We had high schoolers came in and they could do it successfully at first try. You could leave your bacterial RNA at RT for a month without degradation because there is no more endogenous RNase contamination. Since the development of AquaRNA, we don't have to deal with phenol/chloroform anymore, we could just use regular diH2O, regular pipette tips, regular microfuge tubes, and never see our bacterial RNA got degraded. RNA is really tough if there is no endogenous RNase contamination.

I don't know about the rest of the forum readers, but I'm a bit tired of the advertisements.

regarding cells, it's written that the trizol amount used should be 10times the volumes of the cells.
but bacteria wall is quite hard to break. So i would suggest to use 1ml at least and in first approach 1 ml per 50µl bacterial pellet.

Ok, thank you for your answers.
I will decrease the volumes and see what append.

For the moment, there is something wrong with the absorbance ratio:

260/280 : ~1.5
260/230 : ~0.7

To you know what kind of contamination it would be?
I have used 5 mL of Trizol for 2 mL of bacterial culture @ OD600 ~1.

BTW, I have also bought RNAaqua. I will test it in the following days.

c u!