My research group has samples of plant tissue that were pulverized in ln2 using a mortar and pestle before being weighed into tubes and stored in RNAlater at -20 degrees for a few months before processing.
We are now ready to perform RNA extractions and have been running into a few issues.
The first issue is in recovering the tissue from RNAlater solution. After thawing the sample tubes, we have been attempting to centrifuge them to collect the tissue at the bottom and remove the supernatant, but instead the sample "splits" with some going to the bottom of the tube and some staying floating on top. Does anyone know of a high-throughput technique we can use to "filter" tissue from the storage solution so that it is all retained?
A second issue is with RNA yield from the stored tissue. We have attempted a couple of practice extractions using a phenol/chloroform method, but have only obtained very low yields. I have not found a protocol online that involves storing powdered tissue in RNAlater, as it seems it is intended for storing whole tissue. Does anyone have experience with this approach?
Is it possible that a large proportion of the nucleic acids are being released into the solution and are being removed as we filter it out? Are there any other tips you would recommend trying? I have read that washing tissue with PBS after removing RNAlater, but before adding lysis buffer could improve yields from cell cultures. Could this work for tissue samples also?
Another wrinkle in our workflow is that we are dealing with resinous tissue with a high a phenolic content (hemp/hop floral tissue). Column prep kits have evidently performed poorly for processing this tissue in the past, hence the phenol/chloroform protocol we are following. Does anyone have experience with nucleic acid extractions for these tissue types and can recommend modifications that might help us out?
Any and all suggestions are appreciated.
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