[malachig]

Examples of Agilent 2100 Traces (electropherograms)

Here are some examples of Agilent 2100 electropherograms (RNA Nano 6000 assay) for human total RNAs of widely varying quality. RIN numbers reported in the examples range from 1.1 to 10.0. Each total RNA sample was isolated from cultured cells, fresh frozen tissues, or FFPE tissues (indicated in the first column). I would agree with larissa that a three peak trace would be an unusual manifestation of RNA degradation. Having said that, the attached contains many examples RNA qualities from complete degradation to perfectly intact and they can look quite different... Two traces with the same RIN number can look quite different but as the quality and RIN gets higher the consistency improves...

[pmiguel]

Hi,
Can anyone comment on what viral RNA would look like using the Bioanalyzer? Obviously you would not see rRNA peaks unless they were carry-over from the cells the virus was grown in?

Thanks

It would depend on the size of the viral genome you had isolated and whether it was single stranded or double stranded. You might also see sub-genomic processed RNAs that were derivatives of the original RNA genome.

As to the rest of the thread, I would emphasize one issue common for insect rRNAs. The 28s rRNA often is cleaved into 2 parts that co-migrate with the 18s rRNA.

Yes, that is right, a single rRNA peak is often the norm for insect total RNA!

The pdf referenced earlier in the thread mentions this (in a footnote to table 1) is the case for drosophila, but does not show actual traces of this phenomenon. Probably needless to say that no valid RIN score is calculated under these circumstances.

I am not a big fan of the "RIN" score anyway. Since Agilent does not reveal the details of its calculation, it should be regarded as "opinion" rather than data. Also it has been my observation that metrics of this sort tend to be used to avoid having to think about data. Another example is the 260/280 absorbance ratio used in UV spectrophotometry of nucleic acid samples. Here, at least, we know the exactly how the metric is calculated. However its caveats are so legion that its use should be allowed only by licensed practitioners of UV spec.

In both cases (RIN and 260/280) the metrics may well be correlated with sample quality. But viewed out of context they are nearly useless.

--
Phillip

[anneb]

Insect RINs

In case of drosophila, 2 close peaks at the 18 S and one smaller 28 S peak.

Does anybody have a sample electropherogram from an insect (drosophila or, ideally, silkworm RNA samples)? Our silkworm electropherogram has a double peak where the 18S is and almost no peak at 28S resulting in low RIN.

I found a publication online by Aligent (link is below). It says under Table 1 "Drosophila 28S rRNA is split in 2 fragments, comigrating with 18S rRNA." However, I was unable to find an actual electropherogram to compare to my silkworm results.

http://www.chem.agilent.com/Library/applications/5989-1086EN.pdf

Thanks!

[gogreen]

I've never isolated RNA from silkworm, but my best guess is that it might be similar to the drosophila RNA. I'm sorry that I don't have any RNA profiles in my current lab. But a google search got me this one.

http://www.ub.edu/epidd/arrays/protocols.php

also the following article says that if you heat the insect RNA, it looks like a twin 18S peak, otherwise it looks normal. I have not tried them on the bioanalyzer without heating.

http://www.insectscience.org/10.159/i1536-2442-10-159.pdf

[NextGenSeq]

NuGEN has an FFPE RNA-Seq kit which works with much more degraded samples than you have.

[virusHIV]

NuGEN has an FFPE RNA-Seq kit which works with much more degraded samples than you have.

Epicentre also sells one kit for that:
QuickExtract™ FFPE RNA Extraction Kit

[geneart]

miRNA from exosomes bioanalyser

Hi, Has anyone worked on using exosomal RNA on bioanalyser? I have RNA extracted from the exosomes using trizol and bioanalyser was used to check the quality, I do have a good peak at approx.25nt .My question is in the gel pic I see for some samples a band about 200nt and some samples have a band at about 25nt. Also I have just one single peak at the mentioned size in the electropherogram. The ladder looks good. RIN numbers are very low ranging from 0 to 3. Because these are samples from exosomes things already might be chopped off or degraded so I am not bothered much about the RIN numbers. But what bothers me is that larger size band. Has anyone seen anything like this? Please any suggestion is welcome.
Thanks
Geneart

[pmiguel]

If you used the bioanalyzer, please post the trace. Otherwise it is very difficult to assess your situation. You should also go into some detail as to the assay you are planning to undertake with these RNAs. Different assays will have different specifications for input material.

--
Phillip

[SEQquestions]

Geneart - did you do any RNAseq with your samples? We have extracted some RNA and it is giving a RIN of 3. We plan to use RiboZero and then ScriptSeq but also wanted to use a portion for smallRNAseq. I have heard that qtPCR of house keeping genes is a better measure or sample quality than RIN (especially in our case, where we are not simply focusing on polyA+).

If anyone has any experience to share working with such low RIN samples it would be much appreciated

[Oyster_lab]

Hi,

It was relieving to find this forum. I just had my samples ran on Bioloanalizer and they look exactly as you have described (and how it is in the article posted): group of small peaks until reaching the 18S RNA peak and no peak for the 28S. Total RNA samples are from oysters, which are closely related to insects. So, I am assuming their RNA profiles should be very similar. Do you have any update info on this situation?
Any help would be much appreciated.

[Diptipitta]

Hello, I am new to RNA extraction. I am working with rumen (gut) samples. I used hot acidified phenol-chloroform assay for RNA extraction. Here is a gel attached. I am not sure what the peak at 35s represents. RIN values ranged between 3-5.Does these graphs represent degraded stuff or some contaminants.

[kerplunk412]

I don't think your attachment worked.