Total RNA quality (Bioanalyzer trace attached)

peromhc · 02-03-2010, 12:35 PM

Hi All,

Does anybody care to comment on the relationship between total RNA quality and sequence quality (specifically Illumina RNA-seq). I have a couple of irreplaceable samples from wild animals in South America that were thawed and exposed to some hot temperatures accidentally. I have a bunch of inter-region signal that I am worried about.. see attached.

Anybody start out with worries re: RNA quality, then get great sequences, or vice versa??

thanks

shurjo · 02-03-2010, 05:09 PM

IMHO, you're getting a bit too much degradation in your second sample, but then it really depends on the purpose of your experiment. For gene expression differences, I would be uncomfortable using this RNA. For getting an overall look at the transcriptome without being specifically interested in rare transcripts and such, I would go ahead.

Xi Wang · 02-03-2010, 09:57 PM

Hi, could someone show me the Total RNA size distribution without degradation? I have no idea of this kind of plot. Thanks!

jcotney · 02-25-2010, 07:44 AM

Here is a good example of intact total RNA with high RIN value.

Xi Wang · 02-25-2010, 07:59 AM

Quote:

Originally Posted by jcotney

Here is a good example of intact total RNA with high RIN value.

Thanks. And excuse me, RIN strands for what? I am not familiar with such kind of concept.

jcotney · 02-25-2010, 08:18 AM

I believe RIN stands for "RNA Integrity Number". It is a measurement developed by Agilent for their bioanalyzer that takes into account the values for 28S and 18S ribosomal RNAs compared to one another as well as the total signal for the whole trace. We typically do not sequence anything with an RIN value less than 8.

Xi Wang · 02-25-2010, 08:27 AM

Quote:

Originally Posted by jcotney

I believe RIN stands for "RNA Integrity Number". It is a measurement developed by Agilent for their bioanalyzer that takes into account the values for 28S and 18S ribosomal RNAs compared to one another as well as the total signal for the whole trace. We typically do not sequence anything with an RIN value less than 8.

Thanks.

In the figure you attached, it writes: RNA Integrity Number (RIN): 9.5(B.02.07), what's the meaning?

jcotney · 02-25-2010, 08:32 AM

B.02.07 is the current version of the Agilent 2100 expert software that runs the bioanalyzer.

gogreen · 03-09-2010, 12:24 AM

Hi peromhc,
Just got curious about the 3 peaks that you see in the profile. What is this organism??

Xi Wang · 03-09-2010, 05:42 PM

I guess the three peaks are for microRNA, small subunit rRNA, and large subunit rRNA.

gogreen · 03-09-2010, 11:48 PM

Hi Xi wang, I'm sorry if my question was confusing. I meant about the 3 large rRNA subunits. Normally you see 2 of them for mammals and bacteria (In case of drosophila, 2 close peaks at the 18 S and one smaller 28 S peak..I hope I'm right about larger the S unit size). but this profile had 3 distinct peaks starting from the 18S rRNA. That is what I got curious about!

Xi Wang · 03-10-2010, 01:11 AM

Quote:

Originally Posted by gogreen

Hi Xi wang, I'm sorry if my question was confusing. I meant about the 3 large rRNA subunits. Normally you see 2 of them for mammals and bacteria (In case of drosophila, 2 close peaks at the 18 S and one smaller 28 S peak..I hope I'm right about larger the S unit size). but this profile had 3 distinct peaks starting from the 18S rRNA. That is what I got curious about!

I see. Just because there two figures involved in this thread, which makes me confused, I just noticed the last figure. Sorry, I really don't know why there are three peaks..

peromhc · 03-12-2010, 11:09 PM

this was a rodent, but I suspect that the 3rd peak has more to do with RNA degradation than anything else..

larissa · 03-16-2010, 12:21 PM

Hello peromhc, I do not have much experience with the sequencing part but feel comfortable in commenting on the bioanalyzer trace. I would not expect such a sharp peak as result of degradation. Degradation generally would lead to a more widespread increase of the baseline. More than the two major rRNA peaks are very common with plant total RNA profiles. Not much experience with animals but the link below may help, look at the trout profile they show there. You may want to talk to the Ambion experts in tech support. They were really helpful in the past to me. As you RIN is 8, you may still have some good chances of going forward with the sequencing, pending on your objectives. Hope this helps!
http://www.chem.agilent.com/Library/...989-1086EN.pdf

chrisaw01 · 10-20-2010, 10:01 AM

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

malachig · 10-20-2010, 05:21 PM

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 · 10-25-2010, 05:53 AM

Quote:

Originally Posted by chrisaw01

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 · 09-27-2011, 12:50 PM

Quote:

Originally Posted by gogreen

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/...989-1086EN.pdf

Thanks!

gogreen · 09-27-2011, 01:09 PM

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/...442-10-159.pdf

NextGenSeq · 09-28-2011, 07:17 AM

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

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02-03-2010, 09:57 PM	#3
Xi Wang Senior Member Location: MDC, Berlin, Germany Join Date: Oct 2009 Posts: 317	Hi, could someone show me the Total RNA size distribution without degradation? I have no idea of this kind of plot. Thanks! __________________ Xi Wang

03-09-2010, 05:42 PM	#10
Xi Wang Senior Member Location: MDC, Berlin, Germany Join Date: Oct 2009 Posts: 317	I guess the three peaks are for microRNA, small subunit rRNA, and large subunit rRNA. __________________ Xi Wang

02-03-2010, 05:09 PM	#2
shurjo Senior Member Location: Rockville, MD Join Date: Jan 2009 Posts: 126	IMHO, you're getting a bit too much degradation in your second sample, but then it really depends on the purpose of your experiment. For gene expression differences, I would be uncomfortable using this RNA. For getting an overall look at the transcriptome without being specifically interested in rare transcripts and such, I would go ahead.

02-25-2010, 08:18 AM	#6
jcotney Junior Member Location: New Haven Join Date: Feb 2010 Posts: 3	I believe RIN stands for "RNA Integrity Number". It is a measurement developed by Agilent for their bioanalyzer that takes into account the values for 28S and 18S ribosomal RNAs compared to one another as well as the total signal for the whole trace. We typically do not sequence anything with an RIN value less than 8.

02-25-2010, 08:32 AM	#8
jcotney Junior Member Location: New Haven Join Date: Feb 2010 Posts: 3	B.02.07 is the current version of the Agilent 2100 expert software that runs the bioanalyzer.

03-09-2010, 12:24 AM	#9
gogreen Member Location: Europe Join Date: Apr 2009 Posts: 18	Hi peromhc, Just got curious about the 3 peaks that you see in the profile. What is this organism??

03-09-2010, 11:48 PM	#11
gogreen Member Location: Europe Join Date: Apr 2009 Posts: 18	Hi Xi wang, I'm sorry if my question was confusing. I meant about the 3 large rRNA subunits. Normally you see 2 of them for mammals and bacteria (In case of drosophila, 2 close peaks at the 18 S and one smaller 28 S peak..I hope I'm right about larger the S unit size). but this profile had 3 distinct peaks starting from the 18S rRNA. That is what I got curious about!

03-12-2010, 11:09 PM	#13
peromhc Senior Member Location: Durham, NH Join Date: Sep 2009 Posts: 108	this was a rodent, but I suspect that the 3rd peak has more to do with RNA degradation than anything else..

03-16-2010, 12:21 PM	#14
larissa Member Location: NC Join Date: Mar 2010 Posts: 15	Hello peromhc, I do not have much experience with the sequencing part but feel comfortable in commenting on the bioanalyzer trace. I would not expect such a sharp peak as result of degradation. Degradation generally would lead to a more widespread increase of the baseline. More than the two major rRNA peaks are very common with plant total RNA profiles. Not much experience with animals but the link below may help, look at the trout profile they show there. You may want to talk to the Ambion experts in tech support. They were really helpful in the past to me. As you RIN is 8, you may still have some good chances of going forward with the sequencing, pending on your objectives. Hope this helps! http://www.chem.agilent.com/Library/...989-1086EN.pdf

10-20-2010, 10:01 AM	#15
chrisaw01 Junior Member Location: Maryland Join Date: Oct 2010 Posts: 6	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

09-27-2011, 01:09 PM	#19
gogreen Member Location: Europe Join Date: Apr 2009 Posts: 18	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/...442-10-159.pdf

09-28-2011, 07:17 AM	#20
NextGenSeq Senior Member Location: USA Join Date: Apr 2009 Posts: 482	NuGEN has an FFPE RNA-Seq kit which works with much more degraded samples than you have.