Can anyone share their experiences with extracting protein (for IP/WB) with tissues stored in RNALater? On the product website, RNALater is supposed to preserve proteins as well as RNA.
I ask this since we have noticed proteins stored in RNALater seem more degraded than other tissues that were snap-frozen liquid nitrogen and processed.
The following image is from a stain-free gel on the Bio-Rad Chemidoc. We did this as we tried to understand why one student's blots (RNAlater hearts) were suboptimal compared to snap frozen ones.
There was an old thread about this on another forum: http://www.protocol-online.org/biology-forums/posts/37801.html None of the Ambion links work anymore, however.
In general, it seems like there should not be a problem. Looking at your results, you can see some lane width problems. Often this comes from varying salt concentrations from sample to sample. The hypotonic samples will give wide lanes and the hypertonic ones narrow lanes, if I remember correctly. To get all of your samples in the same buffer, then, you might need to perform a TCA precipitation. Alternatively, if you really need to preserve RNA and protein in the same solution, you could store your tissue extracts in TRIzol and follow the RNA and protein extraction protocols here: http://www.ncbi.nlm.nih.gov/pubmed/17489233
Of course TRIzol is organic/dangerous, so it would be nice if there was an alternative protocol using the MasterPure reagents, for example.
There was an old thread about this on another forum: http://www.protocol-online.org/biology-forums/posts/37801.html None of the Ambion links work anymore, however.
In general, it seems like there should not be a problem. Looking at your results, you can see some lane width problems. Often this comes from varying salt concentrations from sample to sample. The hypotonic samples will give wide lanes and the hypertonic ones narrow lanes, if I remember correctly. To get all of your samples in the same buffer, then, you might need to perform a TCA precipitation. Alternatively, if you really need to preserve RNA and protein in the same solution, you could store your tissue extracts in TRIzol and follow the RNA and protein extraction protocols here: http://www.ncbi.nlm.nih.gov/pubmed/17489233
Of course TRIzol is organic/dangerous, so it would be nice if there was an alternative protocol using the MasterPure reagents, for example.
There should not be a difference in protein quality between RNA later tissue and tissue preserved/extracted using other methods. I have compared results from RNAlater preserved mouse lung tissue vs. freshly isolated immediately extracted lung tissue and observed neglible differences in protein profiles (total protein membrane stain) and in immunoblotting results for non-phosphorylated proteins. I have not probed for phospho-proteins so I cannot guarantee that phosphorylation will be preserved but I imagine since RNAlater is denaturing everything, phosphatases should be inactivated as well.
Actually we observed more efficient homogenization/extraction (using a bead-based homogenizer and 1 mL extraction buffer with Roche Mini Complete PI Cocktail) from RNAlater lungs vs fresh lung tissue. We assumed this was due to RNAlater dehydration of the tissue but do not know for sure.
Possible things that could explain protein degradation of samples include the following:
Suboptimal volume of RNAlater vs size of tissue
Not ensuring tissue is fully submerged in RNAlater
Not immediately placing isolated tissue in RNAlater
Not storing tissue in RNAlater at 4oC overnight prior to freezing tissue
Storing tissues in RNAlater at inappropriate temperatures for prolonged periods of time
Too much RNAlater carryover in downstream applications. (we used 1 mL extraction buffer to dilute any residual RNA that we were not able to remove be dabbing the tissue on filter paper repeatedly)
Thank you very much for your well thought out answers..
Mark: Yes, I went to that forum first and since it had limited information and was quite dated, I felt that this question warranted a refresh! Thanks for the lane width information, good thing to learn!
Brian: It is great to hear that RNAlater should not compromise proteins in tissues for WB and to hear that your results have been sound. I am aware of the precautions to take in preparing an RNALater tissue sample for storage but will take better care in ensuring optimal volume of RNAlater and also to rinse it off prior to homogenising in RIPA (of which I did not do).
Sidenote: I've noticed that there seems to be residual protein stuck on the top of my gel in the wells of muscle and pancreatic samples. The heart tissue was homogenised in a glass dounce while the rest was homogenised with LN2 with mortar and pestle. Speaking to a few colleagues isolating mitochondria for native gels, they noticed that suboptimal lysis of protein can show as "smeared" bands.
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