Strange peak in total RNA extractions from brain tissue

[guz]

We have extracted total RNA from frozen brain tissue. To extract RNA, we homogenized 30-50mg tissue with Trizol then added chloroform for phase separation and lastly did a buffer exchange using the miRNeasy kit. We did not perform DNase treatment. We are seeing a large peak at around 100-150nt from the bioanalyzer for all 40 brain samples we used regardless of RNA integrity numbers (see attachment). In fact most RINs are high. Has anyone else seen this? Is this degraded RNA? If it was degraded RNA, wouldn't it be a smear? Or is it possible it is DNA?

[pmiguel]

Those are your small RNAs. tRNA, 5S, 5.8S, etc. Many RNA prep kits do not efficiently isolate them, but Trizol can -- depending on your post-extraction concentration/purificiation method. "miRNeasy" would stand for "micro RNA easy". So one would expect lots of small RNA there.

In some samples isolated with Trizol we see small RNA peaks so high that the BioAnalyzer software balks and refuses to give a RIN until you suppress certain errors it generates.

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Phillip

[guz]

Is this peak specific to brain or do you see it across tissue types when using Trizol?

I wasn't certain this peak was attributed to small RNA species like pri-miRNA or tRNA because I didn't think they wouldn't this abundant. Also, pri-miRNAs are 70nt and tRNA are in the 70-90nt range. Do you think it's mostly 5S, 5.8S rRNA?

Thanks for the help Phillip!

-Andy

[pmiguel]

Well, I can't rule out your peak being something else -- something brain-specific, or even degradation products. But to me it just looks like normal RNA with lots of small RNA in it. However it is a little strange that there is nothing from 25-100 nt. Is it possible that this is something like the "anti-miRNA-enriched" fraction of your RNA?

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Phillip

[guz]

I know there is miRNA present bc I've done qPCR on specific targets. I'm pretty sure you nailed with it by saying it is 5S, 5.8S and degraded RNA.

Thanks for your help

-Andy

[nofchi]

Bioanalyzer band shift 150 -> 100 nt

Dear all, we encountered shift at the similar band size. We isolated total RNA from neuronal cell culture using combination of QIAzol Lysis reagent (QIAGEN) and Direct-zol RNA MiniPrep (Zymo Research). Quality of the isolated RNA was checked on Expirion (Bio-Rad Laboratories) (direct competitor of Agilent Bioanalyzer). Under control conditions, we have observed relatively defined double-band at ~ 150 nt (green). Upon an experimental treatment the double-band weakened and new broad band with most intensive part at ~ 100nt appeared (orange, see image attached). It was consistent in all cases when treatment was used. Do you have an idea what class of RNAs could be responsible for the shift and why could this shift occur? Thank you for response!

[eingabe_ausgabe]

Interesting that I got the exact peaks / bands from insect fat body tissue using almost the exact same experimental set-up (zymo micro kit & Experion). I have virtually eliminated that this might be degradation, but can't figure out what else it might be other than a high amount of material at the 5S/5.8S/tRNA/miRNA region. Did anyone else get any conclusive answers?

[Olaf Blue]

Have you tried depleting the rRNA using Ribo-Zero or RNAse H methods and seeing if or how many of the small RNAs remain or are eliminated using Bioanalyzer?

[eingabe_ausgabe]

Have you tried depleting the rRNA using Ribo-Zero or RNAse H methods and seeing if or how many of the small RNAs remain or are eliminated using Bioanalyzer?

No and it may be too late for me to do that. I am actually trying to retain the small RNAs and I don't think the presence of the ribosomes is necessarily a problem for my downstream goals. I just didn't expect the peak to be so high! I may need to eliminate the possibility that incomplete lysis of my tissue may be responsible for any of the height of the peak at this region and not RNA degradation for some other reason, which I am reasonably sure it is not. If it is not incomplete lysis, and degradation isn't seen near the larger ribosomal subunits (which they are not), then it may be a tissue-specific phenomenon.

I am trying to find a source that explains "Insufficient lysis is indicated by accumulation of small RNA around the 5S rRNA peak."

I appreciate anyone's thoughts!

http://www.nature.com/nprot/journal/v11/n8/fig_tab/nprot.2016.090_F2.html

[pmiguel]

So, correct me if I'm inferring the wrong thing, but the figure from the paper you link to is saying that incomplete lysis results in some of the RNA fractionating with debris that is removed from the RNA prep?

Do you think you got sufficient lysis? Insects often have cleavage sites in their large ribosomal sub-unit RNAs that result in cleavage products the same, or nearly the same length as the small ribosomal sub-unit RNA. We don't think twice if we see this result from insect RNA.

But you seemed concerned about differences in the yields of small RNA products. (Or, what I presume are small RNA products. You don't provide sizing information for the bands.) Unfortunately gels only give you so much information. Unless someone who has run a lot of gels on RNA obtained from the fat bodies of insects happens by the forum, you are unlikely to get actionable answers.

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Phillip

[eingabe_ausgabe]

So, correct me if I'm inferring the wrong thing, but the figure from the paper you link to is saying that incomplete lysis results in some of the RNA fractionating with debris that is removed from the RNA prep?

http://www.nature.com/nprot/journal/v11/n8/fig_tab/nprot.2016.090_F2.html

Does "insufficient lysis" mean cells that are only partially lysed such that only the small RNAs are released and the rest is washed away with the cells? My original interpretation was that, perhaps, some of the cells were recalcitrant due to the nature of the tissue and, thus, took longer to break apart and were subject to endogenous RNases for longer periods of time?

Also, yes, well aware of the insect 18S/28S single peak.

Thanks.

[pmiguel]

http://www.nature.com/nprot/journal/...16.090_F2.html

Does "insufficient lysis" mean cells that are only partially lysed such that only the small RNAs are released and the rest is washed away with the cells? My original interpretation was that, perhaps, some of the cells were recalcitrant due to the nature of the tissue and, thus, took longer to break apart and were subject to endogenous RNases for longer periods of time?

Also, yes, well aware of the insect 18S/28S single peak.

Thanks.

Yes, your interpretation is more likely.

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Phillip