Hi,
I was wondering if it is possible to extract RNA from tissues which have been fixed in 95% ethanol and stored at RT.
The RNA would be used in RT-PCR analysis.
If anyone knows a protocol or any kits available to do this I would really appreciate the help.
Thanks in advance
Sarah
Hi Sarah,
If it is fixed with 95% EtOH, try re-hydrating the tissue with series of decreasing concentration of EtOH (say 95%, 70%, 50%, 30%, 10%) prepared using DEPC water and finally in DEPC water for 10 min interval at every step (time can be adjusted with the size of tissue). All steps should be performed at cold condition (an ice bucket will do the purpose). Then go ahead with TriZol method. The yield will be less, but you can fish up some RNAs.
All the best,
Rajkumar
An interesting question! Many moons ago we used to do in-situ RNA hybridisations and the tissue was embedded after dehydrating in alcohol, so probably your RNA will be preserved, though to what quality it's hard to say?Just be aware that too much residual ethanol when you start extracting will prevent the RNA getting into solution, which would adversely affect your purification. A very old method is to grind the tissue in liquid nitrogen and then extract with phenol a couple of times and then Chloroform, then precipitate with either ethanol or isopropanol. I'm afraid I don't have an exact protocol any more, e.g. buffer conditions, but could try and find out if you think that may be helpful to you? We used to work with either fresh tissue, or tissue frozen at -70. For kits probably best to just check with the manufacturers?
I have always heard that RNA can only be obtained from tissue in RNA-later and not from tissue in ethanol.
Certainly the ethanol would need to be removed somehow before extraction?
Thank you everyone for some good advice. i will take this on board when looking for a possible solution.
Hi Sarah,
If it is fixed with 95% EtOH, try re-hydrating the tissue with series of decreasing concentration of EtOH (say 95%, 70%, 50%, 30%, 10%) prepared using DEPC water and finally in DEPC water for 10 min interval at every step (time can be adjusted with the size of tissue). All steps should be performed at cold condition (an ice bucket will do the purpose). Then go ahead with TriZol method. The yield will be less, but you can fish up some RNAs.
All the best,
Rajkumar
How long the tissue was stored at RT is most crucial, if its been sitting at RT for long time, then it will not been possible. Yes, I think otherwise RNA can be extracted. I have used fresh tissue and using liquid nitrogen crushed the tissue followed by RNA extraction by TRIzol. You can try it, drain off the ethanol quickly, use a mortar filled with liquid nitrogen and with the pestle crush tissue. Then you proceed with the RNA extraction technique available to you. Hope you can try this out.
Thanks for the advice Prasad Pethe . The samples have been fixed for over a month.
Sarah, wondering if you ever got an answer to this or attempted to work out a protocol? I am also interested in trying to get RNA from 95% ethanol preserved tissues...
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