I would like to know if my particular mRNA is being transitioned in and out of p bodies under certain conditions. Looking by qPCR I can see clear changes in transcript levels, but I never see protein. Recent studies have suggested specific miRNA inhibition. It's not a real slam dunk as it hasn't been repeated to my knowledge so I am trying to confirm these results, but would like to go one step further and show localization of the transcripts to p bodies if possible under varying conditions. What are my options for achieving this? I have heard that argonaute proteins are good targets for possibly isolating these structures.
Wow, I stumped everyone? Let me rephrase: Has anyone ever immunoprecipitated stress granules/p-bodies, etc, and then isolated the mRNA from them for qPCR? If so what do I need to know?
Trying to isolate RNA granules has proven incredibly frustrating. The standard way to separate things out like polysomes etc (sucrose gradients) tend to destroy stress granules because they are so fibrillar and loosely associated. I have been looking for/trying to do for several years, along with several collaborating labs, and we have not had much success. we can isolate polysomes....and get reactivity to bits and pieces of stress granules and p-bodies, but haven't found any way (yet) to actually get an intact stress granule or p-body from human tissues. We usually look at them by confocal with markers and look at co-associations with specific RNA or other proteins through in situ or co-IP/co-localization. You can also look at associations using fret. In my work I usually use co-IP for a marker like TIA-1 or staufen or XRN1 depending on the granule and then RT-PCR. Our protocols are published for the co-IP methods (do a pubmed for Strong and Volkening as the authors) but you always have to be aware that you will lose RNA depending on how "close" the protein you are IPing is to the RNA, if it is in direct contact, you will be more succesful, if it is a protein in a complex in which some other piece holds the RNA you may not be. The composition of the lysis buffers are extremely important-too much detergent will rip RNA away very easily.
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