I am trying to find out the protocol to extract RNA from the caecum of the chicken. Thinking logically, the condition of the caecum (or even any parts of the intestine) provides a great source of RNase that may lead to rapid RNA degradation. I am wondering whether rinsing away the feces and any substances in the caecum prior to flash freezing will alter the RNA expression profile. But at the same time, I want to remove the possibilities that will make the RNA of the caecum to degrade. What should I do? I will be grateful for any suggestions. Thank you very much!
Dear, your worries in this case are mostly baseless or simply a phobia. From colon we managed to get highly concentrated and pure RNA with good integrity all the times. All you want to clear about the fact that you need RNA actually from caecum cells (intracellular) not from the caecum surfaces for your research. Here you need to worry more about these intracellular RNAses more than the RNA in intestinal stool or in the bacteria of caecum. In brief, when after sacrification, you apply cleaning, fixing and staining solutions (as per your protocol) over caecum parts like Formalin, Ethanol, dye Methylene blue or else to stain, are all known microbicidals and even before that proper wash with saline also minimizing contaminating burden of caecum. Then the successive storage in RNA Later which is a known bacteriostatic and RNA stabilizer makes job easy so kindly forget such tensions. Honestly, the main thing is proper selection of your caecum part in which you are interested and choice of good RNA extraction kit with a pretty much safer & quick protocol. During your RNA extraction when you will homogenize your tissues that would be in a mixture of lysis buffer with RNAse inhibitors so another safe point here. And how to work on RNA safely, you can just view my detailed answer from my answers list, follow that strictly and be happy and relaxed..And remember in RNA work dedication, concentration and hard work matters a lot, no short cuts or get ready to pay the price...
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