RNA extraction from pancreas of ruminants is very difficult due to the existence of abundant RNase. Previously, our research group tried to remove the pancreas from animal immediatley after slaughter and chipped the pancreatic tissue into small pieces, then washed it using RNase free water twice to inhibit endogenic RNase. In addition, we added exogenetic inhibitor (guanidine hydrochloride) of RNase in the process of RNA extraction, but the integrity of RNA remained not good. May I ask some experienced researcher to obtain a better method to solve above problem? Clould you share your perfect method to me?Thanks a lot.
We routinely obtain RNA of RIN 9-10 from animal tissue (mouse brain) by rapidly freezing the tissue, without any unusual reagents or techniques. It is really important to work fast and clean to maximise RNA quality. Before death of the animal, have all required materials (dissection tools, wet ice, dry ice, pre-labelled sample tubes) prepared and within reach to minimise processing time. Pack a stainless steel tray (I use a food storage container with a flat bottom) with wet ice, and flip it upside down into a cooler containing more wet ice. This creates a flat, smooth, ice-cold surface to dissect on.
After death of the animal, conduct dissection as quickly as possible, and work on the cold surface as much as practical. I highly suggest you not waste time washing the sample with RNase-free water; this does not inhibit RNases and won't achieve anything except giving the RNases in the tissue more time to degrade your RNA. Cut the tissue into pieces no bigger than your fingernail, transfer them into thin-walled plastic tubes or containers, and insert them immediately into dry ice to freeze the sample. You can also use a dry ice-ethanol bath, or liquid nitrogen. Putting the samples in a freezer is not fast enough. They need to be small enough and cold enough to freeze within seconds. If at all possible, work with one sample at a time; have all the samples from the first animal frozen down before you euthanise the next animal.
If freezing samples this quickly is not practical for you or you cannot take dry ice or liquid nitrogen into a slaughterhouse, you could also try an RNA protection reagent, which works by precipitating biomolecules rapidly inside the cells. The best known is RNAlater, but there are others that work the same way. Follow the instructions re. tissue sample size and ratio of reagent to tissue, or it will not work properly.
When you are ready to extract RNA, you want to homogenise your tissue samples directly in a phenol based reagent containing chaotropic salts. Do not use an aqueous lysis buffer. Homogenise directly in something like Trizol, Nucleozol, Qiazol, etc. If your samples are in RNAlater or similar, you can thaw them in the fridge and they don't require special handling. If you have snap-frozen fresh tissue, you need to keep it frozen right up until the moment it's homogenised. Take your samples out of the -80 freezer or liquid nitrogen storage and place them in a cooler full of dry ice. Take samples off dry ice one at a time, smack the tubes on the bench so that the sample rattles around loose inside the tube, and drop it into the lysis reagent. Wait about 5 seconds for it to partially thaw, then start homogenising. Work with one sample at a time and only take each sample off dry ice when you're ready to homogenise. Also, since there is so much RNase in pancreas you might benefit from doubling the volume of phenol/GITC reagent - eg if it says to use 500uL for 50mg of tissue, use 1mL instead. Then you can follow the manufacturer's protocol to isolate the RNA.
Good luck :)
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