18 replies to this topic

[FACS_flow]

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

[miRNA man]

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?

[EmilyG]

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

[Gaetanodiitaly]

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

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?

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.








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

[Sharon Pek]

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

[Fizban]

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

[pbadtopo]

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!

[Fizban]

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

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!

Using Trizol LS does not make difference from normal trizol to me. What you say is very interesting. You mean that by using RNeasy columns we can eliminate the 270nm problem and have a trustable quantitation? if yes have you estimated the loss % for the clean up?

[Paul Dominguez]

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.

[comp3v]

Paul Dominguez, on Jun 3 2010, 10:29 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, could you tell more about your impressions - what means "it seems to work"? I am also thinking to try it (although its price markup is pretty high here in Europe...), but not sure if it is worth it for miRNAs, because at the end you got a exosomes pellet which must be extracted the same way (i.e. Trizol etc) - then what is the advantage?

[Paul Dominguez]

I should say I've only ran 8 samples to test if ExoQuick would work. I'm not done testing it out. In general, it causes a greasy pellet to form after the 12hr incubation. From the pellet, I can collet RNA. The RNA content is about equal to that from collecting RNA directly from serum.
There are still a few thing I need to test. I need to see if this is just from exosome or also microparticles; I want to test for exosome markers. I am also still having problems with 260/280 ratio, so I need to try out RNeasy kit with Exoquick and with serum to see how the RNA yield might change.
I've also tried PEG 20000 to cause protein to precipitate from the serum. It seem to also yield about the same amount of RNA.
The 260/280 ratio is beter in ExoQuick than that from serum by far. I get around 1.5 with exoquick compared to 0.8. PEG is around 1.2 260/280 ratio.
My RNA yields are 8-10ng/ul in a 50ul volume.

[Fizban]

Paul Dominguez, on Jun 7 2010, 09:19 PM, said:

I should say I've only ran 8 samples to test if ExoQuick would work. I'm not done testing it out. In general, it causes a greasy pellet to form after the 12hr incubation. From the pellet, I can collet RNA. The RNA content is about equal to that from collecting RNA directly from serum.
There are still a few thing I need to test. I need to see if this is just from exosome or also microparticles; I want to test for exosome markers. I am also still having problems with 260/280 ratio, so I need to try out RNeasy kit with Exoquick and with serum to see how the RNA yield might change.
I've also tried PEG 20000 to cause protein to precipitate from the serum. It seem to also yield about the same amount of RNA.
The 260/280 ratio is beter in ExoQuick than that from serum by far. I get around 1.5 with exoquick compared to 0.8. PEG is around 1.2 260/280 ratio.
My RNA yields are 8-10ng/ul in a 50ul volume.

What about a 270 nm peak? that's what makes nanodrop quantitation useless. Did u check if using the same amount of RNA from different samples u get the same Ct for each miRNA?
I'm a little skeptic about exoquick not beacause i've tried it but because it is not yet clear to me what can be called exosome and what microparticle both in terms of size and markers. which markers are you gonna test?

[comp3v]

Paul Dominguez, on Jun 7 2010, 09:19 PM, said:

I should say I've only ran 8 samples to test if ExoQuick would work. I'm not done testing it out. In general, it causes a greasy pellet to form after the 12hr incubation. From the pellet, I can collet RNA. The RNA content is about equal to that from collecting RNA directly from serum.
There are still a few thing I need to test. I need to see if this is just from exosome or also microparticles; I want to test for exosome markers. I am also still having problems with 260/280 ratio, so I need to try out RNeasy kit with Exoquick and with serum to see how the RNA yield might change.
I've also tried PEG 20000 to cause protein to precipitate from the serum. It seem to also yield about the same amount of RNA.
The 260/280 ratio is beter in ExoQuick than that from serum by far. I get around 1.5 with exoquick compared to 0.8. PEG is around 1.2 260/280 ratio.
My RNA yields are 8-10ng/ul in a 50ul volume.

"From the pellet, I can collect RNA" - just to be sure, could you clarify how you do this? I mean, RNA extraction from the pellet.
and from which volume of serum you get this yield? 400-500ng seems to be not bad, if it is true RNA amount. (How it was determined? could you show the spectrum?)

[Sero]

Hi,

I am going to try the ExoQuick as well and am very intrested in your discussion !

I get NO RNA at all from NSCLC cell lines (ie. in the culture supernatant) or very few using TRIreagent and with abnormal 260/230 as you guys. I thought using a miRNA specific kit (RNeasy/miRvana) would help but seems like not.
I tried to use equal volumes of TRI/Culture Medium as Chen et. Maybe the miRNAs are too "dilluted"...

None of you did these extractions on cell lines ? If yes what yield did you get ? a lab close to mine uses 1L of culture medium to get 1 µg !!!!
Seems weird to me. but the study model is different though.

Waiting for your answers, and to previous questions as well !
I ll try the Exoquick and tell you what I get. Hope to have the sale as you !

cheers.
CP.

[Sero]

Hi again,

Forgot to ask, how much TRIZOL do you guys use to extract from the serum ???