During the course of the cultivation of a Pseudomonas sp. strain on a toluene derivative we observe a strong darkening of the media, supposedly because of polyphenolic (polycatecholic) compounds formed. The same obervation can me made sometimes while bacteria grow on aromatic compounds .
We like to isolate RNA from the culture but obviously the cells and later the RNA strongly sticks to the polyphenolic/dark precipitation in the media. Does anyone has experience with that and a solution for that? (The RNA extraction from cells grown on succinate work perfectly.)
I think that some compounds on media act on the oxidation of RNA, so it is preferable to use some of soluble antioxidant compounds within medium such as ascorbic acid in concentrations that do not damage RNA.
Thank you very much for your answers, as a first step we will add ascorbic acid and I will later give feedback on that.
Thank you for your question and answers. I am working now with phenolic compounds and I need to extract bacterial RNAs from media containing phenolic compounds. Please, Dr Christian Eberlein l would like to know if adding ascorbic acid in the medium before extraction is working well or what protocol are you using. Thank you.
Since I am working with carnivorous plants, which produce a high amount of polyphenols under stress, we had some problems with DNA extractions and PCR.
After some research, I found out some solutions, which may also work for you.
Since you are observing a strong darkening of the media, we can assume that you have oxidized compounds, probably caused by oxidases. If that is the case, you will need to change your extraction method from the beginning.
Some oxidases can rapidly cross-link compounds to RNA or in my case DNA, during the disruption of the cells, so you need to prevent this to happen.
The best way is to use liquid nitrogen before you disrupt the cells, this will inhibit the work of possible oxidases.
Then you could try to adapt protocols for isolation of plant RNA and DNA that are developed to remove polyphenols and other contaminants or prevent oxidations.
Examples: PVP (polyvinylpyrrolidone) (remove polyphenols), beta-mercaptoethanol (reduce oxidation).
I am attaching one protocol as an example, but you can also find several protocols for RNA extraction of plants.
Thank you very much Matheus R. Belisário-Ferrari! I am sorry for the late answer. The advice with liquid nitrogen might really help. At the moment we are working on method paper on how to improve RNA yields from that kind of samples described.
Mamadou Malick Diallo, we tried to use ascorbic acid but it is consumed by the bacterium and preferred over the aromatic substrate. For that it was no option for us. If you like to look at transcriptomics, it might help to grow your bacteria on a rather low concentration of an "easy" substrate until it is consumed and subsequently add the phenolic compound. After short incubation you should see transcriptional regulation caused by the phenol compound but you will have a sample with sufficient biomass and avoid the formation of polyphenols by intermediates of the phenolic compounds degradation. Thanks to Dirk Tischler for this advice!
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