Dear all
I want to extract RNA from small tissue which is usually less than 0.01g.
I have tried trizol and RNA extraction kit, but both of them could not extract enough RNA from tissue sample. Sometimes, I could see a small pellet in the bottom of tube after centrifuge, but the pellet could not dissolve and could not detect any RNA concentration by qubit RNA HS kit.
Why? What should I do to improve this situation?
Thanks a lot
Dear Jian-Liang Chou,
As you are working with low amounts of RNA, try to add glycogen in the precipitation step of trizol method and extend the precipitation time from a few hours to overnight. Additionally, don`t let the pellet dry more than 10-15 min after the final wash to avoid the problems with resuspension.
Truth is it is small quantity of tissue. Or maybe try a kit with low detection levels(make sure tbey do)
10mg is quite small. Recovery of this amount of RNA by precipitation and centrifugation can be difficult and quite variable between samples. I would recommend to use a column kit designed for small quantities of RNA - I have had very good results with Zymo kits and they make an RNA microprep kit which would be ideally suited to this size sample. Because column kits avoid a precipitation step, the recovery is much simpler and less varied.
If you really need to do a precipitation, I agree with the suggestion above to add a co-precipitant (such as glycogen or linear acrylamide) which can make a big difference to recovery of small amounts of RNA. Also try to keep your volumes as low as possible. RNA is not very soluble in alcohol, but the solubility is not zero, so the more dilute the RNA is the lower the percentage recovery will be.
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