High RIN low DV200?? Can anybody shed some light?

[Micro151086]

I've recently sent off E. coli RNA samples for RNA-seq. The company we have used for sequencing have replied and said all samples failed QC due to degradation. I am looking at the Tapestation values and some of these samples have a RIN up to 9.5 but then a DV200 of 25. I believe this is due to the large distinct band at approximately 95 bp- (no smearing!) which I believed were tRNA. I am now unsure as to whether to proceed with sequencing as they cannot guarantee sequencing results. Has anyone had experience of such a contrast in RIN and DV200 numbers previously? And then gone on to successfully perform the sequencing?

[luc]

The presence of a prominent short fragment band indicates that degradation is not the problem. Bacterial RNA-seq obviously will not make use of poly-A enrichment and thus is not very sensitive to degradation anyhow.
We have not run into a 95 nt RNA band before though.
I would suggest to post the tapestation trace here.

[Micro151086]

Thank you for the reply. I've added some images that will hopefully shed some light on the situation. Many thanks!

[UCan'tBcereus]

Hi Micro151086,

it looks like you have a lot of tRNA in your samples. tRNA is usually in that size range.
I have a few questions.

What Extraction method are you using? Some kits have specific modifications you can make to reduce the amounts of tRNAs that make it through the extraction process if you are not iterated in sequencing them.

Did you plan on doing any ribodepletion? If the sequencing company does, what kind of kit will they use for it? Some ribodepletion methods target tRNAs as well as rRNA, so your problems might be solved there.

And What kind of Kit are they using for Library Prep?

There still might be ways to remove these bands before library prep. If your samples are of high enough concentration, then you could do a bead cleanup with AMpure RNAclean beads at a low bead:sample ratio to remove them. Of course this will lead to extra sample handling and possibly some degradation.

The same idea could be applied post cDNA synthesis as well. Most RNASeq Library Prep kits will have cleanups of after cDNA synthesis. You could lower these bead: sample ratios to help remove the cDNA that would be synthesized from the tRNAs. The only potential problem would be if your tRNAs outcompete your mRNAs and most of your cDNA is from tRNA.

Hope this is helpful!