I have been trying to extract RNA from INS-1 cell lines and pancreatic islets using TRizol. TRizol works best with cell lines that extract 1000ng or more of perfectly pure RNA, however, I have issues with islets. Each extraction from islets yields RNA that varies greatly in both quantity and quality. I sometimes get 2ng of extremely low-quality RNA with 100 islets compared to sometimes getting 80ng of clean RNA with 50 islets. Remember that each time the same protocol and chemicals are used, strict precautions are followed. Could someone potentially help in identifying the issue?
Since you get different yields/quality depending on the tissue type, it's likely as issue with the lysis. If you use a large amount of material then the Trizol doesn't fully lyse your sample, so you get lower yields, and the Trizol isn't able to denature all the RNases, so you get lower quality.
Ensure the sample is fully lysed by 1) not using too much material per volume of Trizol, 2) incubating the sample in Trizol for 10 min at RT after shaking the tube, 3) vortexing the sample if you can see pieces of tissue (although this can shear long RNAs), 4) freezing the Trizol sample overnight at -80C can help with lysing too.
Hello Fnu Palwasha,
Isolation of RNA from pancreatic islets is challenging as the tissue contains high levels of RNases. Spontaneous autolysis may also be a problem during extraction of RNA from pancreatic islets.
RNA degradation occurs while the pancreatic tissue is removed during dissection. So dissection has to be performed as quickly as possible.
You may want to refer to the paper mentioned below wherein the investigators assessed different RNA extraction methods from pancreatic tissue with focus on the effect of frozen storage and RNase inhibition strategies, both of which affect RNA quality.
Iran J Med Sci. 2014 May; 39(3):282-288.
PMID: 24850986
Best.
Malcolm Nobre Thanks so much, I am hoping the paper's findings will help.
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