Hi there,
I know that there are very good kits for miRNA isolation, but I want to use Trizol for this purpose. It is known that Trizol selectively loses GC-rich small RNAs when extracting RNA from small starting material, and I guess it may still have this same problem when using larger starting material for RNA isolation (i.e.losing a subset of miRNAs).
Does anyone of you have specific comments on how to successfully and efficiently use Trizol for total RNA isolation which contains small RNAs as well? What modifications should be applied to Trizol isolation so that small RNA could be preserved?
Thank you in advance,
Sharif
Hi Sharif,
I guess you may refer to Narry Kim lab paper (Mol Cell 2012). According to their conclusion, 1) you should have no problem when using sufficient number of cells for extraction, because total RNA functions as a kind of 'carrier' for small RNA recovery, and 2) if you are still concerned about biases, you can supplement alcohol precipitation with additional MgCl2.
Trizol is good for total mRNA. Try using ambion kits for miRNA. You will loose alot of material using Trizol, especially if your starting material is low.
Hi, if you use regular filter, called column in commercial kits that is design for total rna, and you will never get srna. I used ambion for sRNA, and it has detail proticol about columnes and their size. Also, qaigen has a good mirna kit. I would read snra kits manual before i decide about trizoL.
As I know trizol give more rna, but stability might be a problem, and srna protocol was a litle bit longer than standard total rna isolation.
I hope it helps. Best luck...
Hi Sharif
Our lab routinely uses Qiazol for miRNA isolation. We uses Isopropanol for precipitation in -80 overnight. This provides great results for us.
Best of luck
Hi Lachlan,
Thanks for your input. Could you please explain a bit more? As I said, for some reasons, I do not want to use any kit, do you use Qiazol in a kit or what?
Sharif
Sharif,
Qiazol is the Qiagen branded trizol. Just follow the protocol that comes with the product. Make sure your precipitation stage is at -80 degrees Celsius overnight.
Regards
To be clear, we don't use any other kit. We just complete a standard RNA precipitation using the Trizol reagent. We just ensure that during the precipitation stage we use Isopropanol and leave it at -80 as I said before. We use the miscript II for cDNA synthesis.
Hi Dooyoung,
Thank you for your comment. What is the function of MgCl2 in this setting? Do you have a reference article for its use (or probably a personal experience)?
Sharif
Does anyone of you know what the function of MgCl2 is in RNA isolation, as suggested by Dooyoung Lee (see above)?
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA