Agilent Bioanalyzer: Unexpected signal in 5S region

[juelraja]

Hi guys!

I keep having this problem with zebrafish RNA isolated with Tri Reagent. On nanodrop everything seems to be OK (260/280 ratio ~2 and 260/230 ratio >2).

Do you have any idea what can cause this unexpected signal and how to get rid of it? Can I expect this to do harm later on with RT-qPCR?

Electropherogram seems quite nice but RIN values cannot be calculated.. (Hopefully managed to upload a picture)

[juelraja]

Hi,

I got rid of the unexpected signal in most samples by denaturating the samples in 70C for 2min (as suggested in protocol)...

[pmiguel]

Sometimes with Trizol samples you see enormous small RNA peaks that freak out the Agilent software. Nothing wrong with the samples though. They just produced a high yield of small RNA. In your case I don't see anything wrong with your chromatograms. That means the software produced an error that it should not have.

What we do is find the parameter setting (in the pane on the right side of the the 2100 Expert software window) that deals with the threshold for giving an "unexpected signal in the 5S region" and increase it until it removes the error.

[juelraja]

Thanks a lot for your answer pmiguel!

Do you know if this observation is reported in literature? Is this related to zebrafish RNA or do you find similar problems with other samples also?

-Juho

[pmiguel]

Thanks a lot for your answer pmiguel!

Do you know if this observation is reported in literature? Is this related to zebrafish RNA or do you find similar problems with other samples also?

-Juho

I don't recall any mention of it in the literature. It is a goofy interaction between Trizol (acid phenol) RNA preps and the Bioanalyzer 2100 expert software.

Actually problems of this sort, Agilent's tech support is pretty good with. They can tell you which parameter you need to alter to get the error to go away.

The other samples I saw this with, did happen to be fish, although not zebrafish. But I really think it is just a matter of the efficiency of the clean-up methodology retaining small RNAs. If it is good, you get giant small RNA peaks.

That said, your small RNA peaks did not look large to me. So, I don't know what the deal was there.

--
Phillip

[mchekula]

Hi, I have a large peak in 5S region, larger than rRNA peaks, but RIN calculated based on 28S/18S ratio looks OK (8-8.5). RNA isolated TRIfast reagent. Any idea what it could be? Thanks!

[pmiguel]

Just looking at this ancient thread that mcheckula revived. The answer I gave in 2013 misses the actual issue here. The "signal in the fast region" is the actual 25 nt standard peak. The software has incorrectly identified peaks inside the 5S region as being the 25 nt standard. So the best fix is to set the real 25 nt peak as the lower size marker.
--
Phillip

[mchekula]

In my case it's not 25nt peak though. Any opinions on the attached plot?

[pmiguel]

Hi, I have a large peak in 5S region, larger than rRNA peaks, but RIN calculated based on 28S/18S ratio looks OK (8-8.5). RNA isolated TRIfast reagent. Any idea what it could be? Thanks!

Most likely it is a mixture of small RNAs -- 5S, tRNA, etc. "TRIfast" I have not heard of, but I presume it is another version of "Trizol", the commercial acid phenol reagents. For whatever reason, acid phenol methods seem to be extremely efficient at retaining low molecular weight RNAs that other methods mostly do not retain.

That said, I don't know that I've ever seen the 5S, etc. peak that large. So there might be something else going on.

--
Phillip

[mchekula]

Trifast is an analog of Trizol indeed. Yes, the height of the 5S/short RNA peak in comparision with rRNAs troubles me too - cant imagine any combination of naturally occuring short RNAs make such a large peak. But can it be degradation?

[cement_head]

Looks pretty good. Likely won't interfere with anything, including NGS. If you are concerned, you could try a bead clean-up (Promega ProNex), but I personally wouldn't.

[mchekula]

Looks pretty good. Likely won't interfere with anything, including NGS. If you are concerned, you could try a bead clean-up (Promega ProNex), but I personally wouldn't.

so you would fully rely on RIN? What do think is the source of the 5S peak, if not degradation?

[pmiguel]

You would probably need to sequence it to find out what it is. But my guess would be lots of tRNA, or some other small RNA. Or something unexpected.

Since the rRNA peaks appear to be intact, it seems unlikely your RNA is degraded.

--
Phillip

[cement_head]

so you would fully rely on RIN? What do think is the source of the 5S peak, if not degradation?

No, I do not rely on RIN, but I analyse a lot of non-model critters. I usually go by 28S vs 18S peaks and the gel.

[mchekula]

You would probably need to sequence it to find out what it is.
Phillip

good idea, thank you