I am doing a dissection to get a small amount of tissue for RNA isolation. The saline was made using non RNase-free/sterile parts. Also, the area I am dissecting is very close to the gut which occasionally becomes permeated so there is a likelihood of high levels of RNases during the dissection.
Somebody suggested I add BME to the saline to inhibit RNases. My question is what would be a sufficient amount to add to inhibit RNases without making it immediately toxic to the tissue I'm dissecting.
The suggestion is good, BME is a reducing chemical and kills RNases (denatures them). You can use DTT instead Article Replacing β-mercaptoethanol in RNA extractions
As for the animal -since it is being dissected, it must be dead so there should be no impact. However, when a cage with 4-5 mice is brought to the lab for dissection, it is recommended to keep the cage inside the fume hood to eliminate the stench; this will also address your BME problem (impact on other people in the same room). The other animals should not see the dissection procedure as it makes them stressed. Therefore, covering the cage with cloth is one way. To further address this, it is recommended to keep them away from the dissection area because they smell blood and again it makes them stressed. You can google these recommendations
For maximising RNA quality from fresh tissue, the single most important thing you can do is keep it really cold. Whether you decide to use supplements like BME or not, do your dissections in ice-cold buffer or on a chilled metal platform (I use a stainless steel lunch box packed with ice slurry - I flip it upside down on the bench and use the underside as my dissecting platform.) For my tissue type (mouse brain) we have found that adding RNase inhibitors helps a lot. 1uL/mL of 1M DTT and 1uL/mL of RNaseOUT added to the buffer for dissection/storage, combined with keeping everything ice cold, lets us get high quality RNA (RIN 8+) sometimes after half an hour or more of handling and dissection steps between death of the animal and beginning RNA extraction.
Laura Leighton Hi Laura, thank you so much for this! I may instead choose to go with DTT if you have found luck with it. I will be doing my dissection as cold as possible. The cold metal dissection stage is a great idea!
I was originally going to attempt to snap freeze my tissue without submerging it in a liquid, but I may choose to freeze it in the dissection saline with these components added.
Naya McCartney I think the RNaseOUT has a much bigger effect than the DTT - the DTT is just in there because most protein-based RNase inhibitors (I think all except Superasin) require reducing conditions and are essentially useless unless DTT is added.
I would recommend freezing your tissue samples without liquid, or with the smallest practical volume (eg if you have pelleted cells). The liquid with RNase inhibitors will not provide any extra protection for frozen samples, and it will interfere with RNA extraction. I normally drop frozen tissue samples into a dounce homogeniser containing a chaotropic RNA isolation reagent (nucleozol or trizol) and homogenise them without them ever having thawed - this makes a huge difference to RNA quality. It would be much more difficult and perhaps impossible to do this with tissue samples frozen in a block of ice, and then you would also have too much water in your samples.
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