I want to check the expression some bacterial genes in planta condition by qPCR. I am looking for protocols to extract the bacterial RNA from rice roots although it seems a technical challenge. If you have experience or comments on this step, please share it with me.
Thank you in advance.
Read this article. This may be helpful for your study.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4071639/pdf/fmicb-05-00286.pdf
I've experience on coffee RNA extraction, roots and other tissues. I never extract bacterial RNA in roots, but the extraction of coffee is already complex.
I used to do a better cleaning with a SDS buffer in water bath ... from there I centrifuge, collected the supernatant, made a chloroform and began the extraction with Trizol. At other time I did chloroform after the water bath (the material was dirtier), followed by Trizol.
I imagine that the amount of RNA is a problem, especially if you'll do qPCR two-steps. In this case, I recommend to use one-step (you can start your qPCR with 25ng RNA). See more in: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2291734/pdf/0160266.pdf
Sorry about my english!
Hi Lucia,
Thank you for sharing your experience. I also get some advice that I might get very low concentration of bacterial RNA to be able to proceed further steps (such as qPCR). That is why I think I should ask around for comments and experience.
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