totalRNA isolation for miRNA

[Ironman]

Hello everyone,

I am working with the TriZol method and need some tips on totalRNA isolation from small quantities of cells.
I discussed this topic with a company were are collaborating with and their technician advised me to
use TriZol instead of any kits cause TriZol gets the most out of each sample.

Anyhow, after having done only very little RNA isolation so far I must say it is very tricky.
Somehow everyone does it differently. What I am looking for is a good protocol or
tips to extend my protocol for isolating totalRNA from cell numbers as low as
30.000.
A crucial point seems to be the precipitation step of the RNA.
I plan to use the sodium actetate precipiation step of small nucleic acids.
Does anyone have any experience on this?
My problem is that the OD I get is generally bad. It ranges between 1.5 and 1.6 analyzed with a NanoDrop.
Somewhere I read that a low OD is due to Phenol contamination and that you can get rid of this with an
additional chloroform extraction step after having picked the aqueous phase after phase separation.
Can anyone confirm this or is there another way to get a better OD?

I would be grateful for any kind of hints on this!

Cheers
Chris

[Katy_Bobbles]

I can't comment on the sodium acetate but when I did RNA isolation I used isopropanol for the precipitation phase. 0.5ml for every ml of TRI reagent that was used initially. All my 260/280 values were between 1.8 and 2. Your results indicate protein contamination. I isolated RNA from around 12 samples all indicated there was no contamintation with DNA or protein so i would try isopropanol. Also be careful when you precipitate that you don't cause contamination during the phase separation.

Don't know if its any use but as I say it worked for me and helped me get a first class honours degree ha! Sorry couldn't resist the chance to show off my news hehe!

[microRNAmiRNA]

FOR miRNA microRNA ISOLATION SEE PUBLICATION

MicroRNA isolation and stability in stored RNA samples.
Mraz M, Malinova K, Mayer J, Pospisilova S.
Biochem Biophys Res Commun. 2009 Dec 4;390(1):1-4. Epub 2009 Sep 19. Review.