We found out the hard way that RNAlater blocks x-rays, and now we have thousands of 20-40mm long fish that we can't x-ray unless we can leach RNAlater back out of the tissues. But I have no idea if this is possible or what protocol to use. Does anyone have any experience with this?
We understand that we'll lose the ability to do RNA extractions when we do this, so I'll take samples before I start trying to remove anything. But I don't want the fish to rot while I'm working, so I'm not sure just soaking for a few days in distilled water is the way to go.
Any tips would be greatly appreciated!
RNAlater is a concentrated salt solution, so I think you would be able to leach it out by soaking in a less concentrated salt solution. I don't think you would want to use water though, because it's hypo-osmolar relative to the normal contents of fish cells and will result in swelling and lysis of the cells. For mammalian tissue I would use PBS. You probably already know of a buffered salt solution that is isotonic to fish tissue, and that's what you should probably use.
If these were my samples I'd take just a few fish and try to re-equilibrate the salt content by soaking them in isotonic saline for multiple days, with daily (or more frequent) changes of the soaking solution. To reduce the risk of decomposition or bacterial growth I would do it at 4C, and add 0.01% sodium azide to the soaking solution. Sodium azide is EXTREMELY toxic so handle it with care and collect all the solutions for chemwaste disposal. Is setting up your x-ray experiments really difficult and expensive? Maybe you could prepare a few fish soaked for different lengths of time in isotonic solutions and see how long it actually takes to leach out enough of the RNAlater to get the expected results on x-ray?
Laura Leighton , very good suggestion. I tried that last week, soaking for 1-4 days. Unfortunately, none of the times soaked the RNAlater completely back out. Distilled water DID do that, but the fish also rotted (as expected).
But it turns out that the RNAlater was acidic enough that it decalcified the bones, so there's never going to be anything to x-ray. We're seeing of a CT scan works better, but we may just not be able to do vertebral counts on these. But thank you for the help; I think it would have worked for a different salt solution.
Harmony Borchardt-Wier oh no, that's a bummer! This kind of thing seems to happen a lot when trying to get as many measurements from one sample set as possible. I hope you get the data you're hoping for!
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