Total RNA quality (Bioanalyzer trace attached)

[peromhc]

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]

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]

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

[jcotney]

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

[Xi Wang]

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]

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]

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]

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

[gogreen]

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

[Xi Wang]

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

[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!

[Xi Wang]

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]

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

[larissa]

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/applications/5989-1086EN.pdf

[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