We normally isolate RNA from rice and Arabidopsis by using trizol. We would like to do RNA-seq and small-RNA seq. There are commercial kits like miRVana which allow to isolate total RNA including small RNA. Is trizol method good enough or is it recommended to use these kits to have RNA of that quality? If yes, why is this the case? Thanks.
Hi Sachin,
Be careful on what reagent you use for your small RNA extraction. Please see this retracted paper from "Retraction Watch". They used Trizol for their small RNA extraction:
"Group retracts microRNA paper after realizing reagent was skewing results." [ http://retractionwatch.com/2012/07/13/group-retracts-microrna-paper-after-realizing-reagent-was-skewing-results/ ]
Part of authors' response:
"In brief, we found after publishing the original paper that our results were due to a technical problem associated with widely used Trizol reagent. When we used a different protocol (which is infrequently used in the field), we did not observe the same phenomenon. It turned out that small RNAs with low GC contents and/or secondary structure are lost from RNA purification when a small number of cells are used (miR-141 was a typical case). This is not due to a biological regulation, but a technical problem associated with Trizol reagent."
We have used trizol to extract RNA for RNAseq analysis. We had to because our plant species (a cactaceae, rich in polyphenols and polysaccharides) is a tricky one and there was no kit available to extract RNA of good quality (we also had plant tissue limitations, as ours is an endagered species). So, trizol. The extraction was good, i.e., we had clean samples and a good yield; the quality assessment of the reads didn't show a skewed GC content, then, for large RNA molecules you shouldn't be worried about gc bias and you can proceed with the trizol extraction.
As for miRNA extraction, I would go for optimized kits given the reasons highlighted in previous answers in this topic.
Thank you Gustavo and others. Did you check your RNA on bioanalyzer, what RIN did you get? I don't worry for miRNA, as we will send total RNA to the company, they will prep small RNA library for us. So trizol method is unbiased, it extracts all kinds of RNA molecules, unlike some kits which exclude RNA less than 200 bp. My concern with trizol method was purity and quality as determined by bioanalyzer. Also, many people do DNase treatment before RNA-seq, did you do that too, if yes, how? Please reply.
Thank you very much.
I did not check the RIN personally as I don't have access to the BioAnalyzer, but the company that sequenced our samples did. My RIN values were 8.3-9.6, most of the companies will require a RIN >6.5 and a 28S/18S > 1 (but the closer to 2, the better).
The DNAse treatment should be done according to your provider's protocol, so check the specifications for the DNAse you're using (it tipically includes a 30 minute incubation at 37C to digest DNA, then EDTA and heat shock to deactivate the enzyme or an aditional RNA purification step to recover your RNA, DNAse-free).
Also, take a look on this to decide wether or not it is a good idea to prepare your libraries with "total" RNA isolated with trizol: http://www.sciencedirect.com/science/article/pii/S1097276512004819
The paper in the link Gustavp posted was published by the same group who retracted their first paper because of using Trizol. In my personal opinion, it looks like that your downstream experiments are mainly on small RNA sequencing and small RNA library preparation. So, it should be a good idea to use a small-RNA specialty kit for small RNA isolation. This will save you a lot of pain if later you find out that something is wrong after the whole library has been constructed. Just a thought.
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA