totalRNA isolation w/ Trizol for miRNA Array - (Jul/10/2008 )

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

---

you must have got somehting wrong here... you are talking about OD 1.6... are you sure your didn't meant the A260/280 ratio ?

can you tell us what the RNA concentrations are ?

I knew of some people who were able to do a RevTrans-PCR from a RNA trizol extraction from a single C:elegans worm (900 cells) so I guess 30'000 are not too bad...

for the precipitation of RNA, my protocol only makes use of cold isopropanol, it's the basic protocol from GibCO but I know you can make a supplementary wash with dilute ethanol afterward.

if you are afraid of phenol contamination ( also see here) I usggested you make the chloroform wash with a little higher volume of chloroform.

---

When working with very small amount of nucleic acids, precipitation can be done at -20 overnight to have a complete precipitation.
During centrifuging the precipitate can smear on the wall of the eppendorf tube, and does not necessarily slide down to the bottom. So it is generally a good idea – even if you don't see a pellet – to roll a drop of water on the outer side of the eppendorf tube when you try to dissolve the pellet, thus collecting the invisible RNA from the side of the tube.

---

I am sorry yes I meant the A260/280 ratio. It ranges between 1.5 and 1.6. I also got 1.4 once.
People told me that this is not very good, QC at a Bioanalyzer should resolve this but this is still to come.

Concentrations are quite low and around 400ng in the 10ul RNA sample (40ng/ul)
quantified with a NanoDrop.

Thanks for the link I will try that as well. But have you ever heard of an additional chloroform step to get
rid of the phenols?

@Kubac
Thanks for the hint, I will try this on my next isolation.
The Sodium Acetate precipiation requires overnight precipitation as well.

---