miRNA isolation from liquid samples - (Jul/21/2009 )

Hello everybody,

I have been trying to isolate miRNA respectively total RNA from liquid samples (mouse serum)
and I failed a couple of times now (at least that's what I think)
So far I only have expertise in isolating RNA from cells or tissue but never from liquid samples, so
I don't know what the yield and quality will be from let's say 200ul of serum.

So far I have tried the TrizolLS protocol provided by invitrogen with glycogen as a co-precipitant
as well as a modified protocol for liquid samples using Qiagen's miRNeasy column-based method.

For RNA isolated using the Qiagen kit I only get an amount of 3-4 ng/ul with no elevation of the
spectroscopy curve on the NanoDrop at 260nm.

For RNA isolated w/ Trizol i get quite a good yield of about 50-100ng/ul however, organic contamination
is quite high (A230) and what really puzzles me is that the peak of the spectroscopic curve is at around
271nm, which I really do not understand.

So first of all my question is, is this normal? Or what am I to expect when isolating RNA from serum?
And secondly I would really appreciate any input concerning this topic which might help me
isolate high quality RNA from serum.

Thanks a lot!

Chris

I have no experience in isolating from serum...but weren't there a couple of papers last year where they profiled miRNAs from cancer patient serum? Maybe you can follow their protocol, or even try shooting off an email to the PI or grad student/postdoc who published it?

Hi Chris

I was very interested to read your post about miRNA extraction from serum and wondered if you been able to sort out the problems?

I am just starting a project looking at miRNAs in human serum and have used TRI reagent BD (a cheaper version of Trizol designed for blood based samples) to isolate them.
I also get a peak at around 270nm and think this is due to TRI/trizol reagent contamination. Not sure how much of an effect this is going to have on my downstream applications but I'm going to try extra ethanol washes following precipitaion of the RNA to reduce it.
The good news is I have looked at the profile of the RNA extracted and in serum it is all small species - no mRNA detected, therefore no need to enrich for the small stuff and waste precious sample.

I have also tried the mirVana kit from Ambion/ABI which did work quite well but the elution volume (100ul) was too big for my experiments. The ABI rep was very good and gave me a free kit to try so you may be able to do the same thing.

Hope that helps and please let me kow if you have since been able to sort out your 270nm problem!

BW
Emily

EmilyG on Oct 15 2009, 08:40 AM said:

I got ~10ng/ul with the nanodrop reading, with human serum. ~384ng in total, kind of depressing.
Surprisingly got a nice A260/280 of 1.85 A260/230 of 0.2

Any one with similar experience?
I used Qiagen's RNeasy plus kit

I've been working on the 270nm peak for months, without solution. bad news is you cannot rely on nanodrop for RNA quantitation (yes, i'm aware that people DID publish that the can quantitate it but this does NOT apply to me). I've tried EVERY kit and lysis method available (and also still not available) without success. maybe with a bioanalyzer it can be done, i do not own one.
good news: the RNA IS there. speaking about mice if you collect total blood you'll discover that you can use as little as 30ul of plasma with 1ml of trizol and glycogen. do try one real time resuspending pellets with 100ul of water, use 2ul for single specific RT (i use ABI) and tell me.
works the same with 400ul of human plasma but yield is, of course, lower speking of Cts.
hope it helps
Fiz

As I said on other thread (next to this one). The best and easiest way to isolate miRNA from serum/plasma is with Trizol LS. Yes you do get very low 260/230 ratios and a very noticable peakat 270nm on nanodrop. This is probably due to contamination of phenol (Trizol) or more likey other contaminant that absorbs strongly at 270nm. When we have treated samples in parallel serum and cell lines for example you get very clean preps with the cell lines which to me suggests it is a particular problenm of plasma/serum possibly due to high protein/albumin? or bile salts etc. The RNA is there and is perfectly fine for techniques such as Taqman qRT-PCR as generally you would dilute a fair bit (1/30). The problem we found was when you want to do anything with it undiluted (say arrays) then the contaminants appear to inhibit reactions (RT etc.). But you can get over that (and see much nicer spectra in nanodrop) by cleaning up on RNeasy columns (according to their protocol) just make sure you use 80% EtOH for preciptating instead of what they say unless it will all go through in wash!

pbadtopo on May 13 2010, 09:29 PM said:

I've been having the same problem with 260/280 ratio. I've tried Ambion's TRI and miRvan kit without any success.
pbadtopo mentioned the RNease Easy kit will resolve the problem, but the kit doesn't collect miRNAs. By using 80% EtOH, I will retain the miRNA. I don't need to do anything else?

Also, has anyone used ExoQuick ExoQuick from System Biosciences? I'm trying it out and it seems to work, but I'm still skeptical about what I'm getting from the serum.

Paul Dominguez on Jun 3 2010, 10:29 PM said: