Low RIN of plant RNA and high abundaces of rRNA.

[yonne]

Hi there and thanks in advance for your input!

We extracted total RNA from (plant) leaves, using the quiagen RNeasy kit. As expected, we isolated a lot of plastid rRNA that also shows up in the electropherograms from agilent's bioanalyzer out.pdf. IMHO the traces look fine and do not show signs of degradation, but why do we get such low RINs? Could the plastid rRNAs be the reason for that?

We plan to RNAseq those samples (we aim for mRNA) and I was wondering if the high amount of rRNA will interfere with the polyA library prep?

[Raul Llera-Herrera]

maybe the version of the Agilent's software that you used does not consider the complex nature of plant RNA, and is underestimating the quality of the RNA.
look at this link:

http://www.chem.agilent.com/library/applications/5990-8850en.pdf

There is a great work that underestimate low RIN on plant RNA-seq experiments:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0050226

We have had RIN of 2.5, and we hope to have at least a core transcriptome for a microalgae from them. Your samples look very nice.
Good luck!

[yonne]

Your samples look very nice.
Good luck!

thank you

The paper was really helpful! However they do not mention if high amounts of rRNA will lower the yield of mRNA during the polyA fishing. Do you have any ideas/experiences with this?

And I don't think it should be a software problem. We are using the B.02.08 version and complex plant tissues are considered since B.02.07.

[pmiguel]

The problem is your ladder!
See how the sizes of your rRNA peaks are all wrong? (25S rRNA is not 5,000 bases!) This will have been caused by the software mis-identifying one or more of the ladder peaks. (The wrong sizes were assigned to them. )
What needs to be done is to look at the ladder plot inside the Expert 2100 software. This will need to be done on the .xad file. Find the mis-identified size peak. (Compare to another run, if necessary.) Right-click on the upper or lower size marker in the Peak Table or tinker around with the parameters until the software correctly sizes all the peaks.

--
Phillip

[pmiguel]

About amount of mRNA in plant RNA samples. Yes, we have found they tend to give lower mRNA yields. Whether this just means plant rRNA is more abundant than animal rRNA, and/or plant poly A tails are shorter and therefore give worse polyA yields, I don't know.

If you are concerned, you can check your polyA yield. Transfer 10% of your bound polyA on the oligo dT beads to another tube. Elute it using water or buffer instead of that "elute fragment prime" mix (or whatever they call it now). Run an aliquot of this on either a nano or pico chip (depending on what your expected yield is.) Alternatively assay the amount fluorimetrically with a fluor that will detect single stranded polynucleotides.

--
Phillip

[yonne]

Thank you Phillip,

indeed the ladder is mis-identified. As soon as I will get back to the lab I will try to fix this and see how it influences the RIN.

However, in a previous_bioanlyzer_run.pdf (with correct ladder) the traces look alike, but RINs are still not optimal. Probably total leaf RNA is still kind of complex for the software and will end up with lower RINs.?

The same pattern can be seen in the two links Raul Llera-Herrera posted earlier. In Agilent's pdf the sunflower leaf has a complex trace and a low RIN. In the PLOS One paper the leaf RIN average (including 480 samples) is around 6.

[pmiguel]

Yes, the rRNA pattern doesn't look quite normal. Possibly one of the ribosomal RNAs is cleaved in sunflower, resulting is a strange pattern that is getting a lower RIN than it deserves. I recommend just using your best judgement rather than believing the RIN.