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 band at approximately 95 bp- 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 numbers previously? And then gone on to successfully perform the sequencing?
The debate on the usefulness of RIN for assessing RNA integrity is still active Article Measure transcript integrity using RNA-seq data
. Documented limitations of this matric have led to the development of other approaches like DV200 . I will say repeat the RNA extraction and take care to reduce degradation.On a different note, what is your reason for suspecting tRNA as the sole culprit for the spike? I think that is unlikely.
Thank you for your response! My questioning of the relevance of the DV200 for these samples largely stems from comparing my bioanalyser traces to those in the published literature for the same bacterial species. I've included a screen shot of my trace. I would think that if there was an issue with the prominent smaller bp band and the resulting DV200 then this would occur in all cases with large amounts of tRNA or small RNAs. We also performed a Qubit IQ measurement which did not suggest degradation.
Sarah Forbes
I agree that having large amounts of tRNA or small RNAs will decrease the DV200 percentage. However, it is not clear why total RNA will have such distorted proportions except you enriched them.
Though most of your samples have good RIN numbers, some of the low bp bands can cause serious problems with the library preparation and fragment selection.
RNA fragment size is a better determinant of successful and reliable library preparation using standard kits compared to RIN. I suspect the service company you are using applies one of the standard library preparation kits, which may not be suitable for preparing libraries for small RNA sequencing.
If this size behaviour can't be resolved by reducing degradation during RNA extraction, then you need to ask the company for an alternative library kit to suit your needs.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA