Hi,
I am currently working on RNA extraction on blood serum samples using Trizol. However, the RNA yield is quite bad, on average, only yielded 200ng per 200ul of sample input, and the A260/280 only lays around 1.45, and A260/230 only lays around 0.4.
I also used an extraction kit from Qiagen (miRNeasy Serum/Plasma Advanced kit) to extract, and the results are still the same. is it possible that my issue is more on how the serum is prepared than the RNA extraction process?
Has anyone tried the Trizol LS? Will this concentrated Trizol yield better RNA? and is there any modification you guys have tried that can give out a better result?
Thanks!
Peter
Hi Peter Kan, I have used Triazol for RNA extraction from the tissue and in most of cases the yield was really good. With blood, I do not have any experience. However, if you are getting same low yield with two different methods then I think you need to re-check the steps prior to RNA extraction, for example cell lysis, homogenization, purification etc.
For RNA extraction it is ALWAYS recommended to carry out the procedure on ice and the process should be too quick to avoid degradation.
Serum doesn't have a lot of RNA: it's a cell-free biofluid by definition, so all the RNA present will be free-floating RNA, either mRNA released by ruptured cells, or circulatory microRNAs, or mRNA/miRs in exosomes/microvesicles.
200ng from 200ul is pretty good. The reason you're seeing poor 260/230 and 260/280 ratios is because you don't have a lot of RNA, not because it's dirty (these are ratios, after all). You could try to clean it up, but frankly you'll just end up losing material. Probably it's good to go, as is.
I would proceed with a few samples and see if your downstream workflow behaves as expected.
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