Tweet
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?
-
-
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. -
Thank you for the reply. I've added some images that will hopefully shed some light on the situation. Many thanks!Comment
-
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!Comment
-
Proteomics platforms are evolving rapidly, with advances in mass spectrometry and affinity-based approaches expanding what researchers can detect and at what scale. As the field moves toward deeper proteome coverage and clinical applications, scientists face an increasingly complex landscape of tools. This article will explore how researchers are navigating these choices to find the right platform for their work.
The systematic characterization of the human proteome has...07-20-2026, 11:48 AM -
Genomics studies in neuroscience face a special challenge due to the brain’s complexity and scarcity of samples. Mapping changes in cell type and state using conventional next-generation sequencing methods remains challenging. Advances in technologies like single-cell sequencing, spatial transcriptomics, and long-read sequencing have opened the door to deeper studies of the brain and diseases like Alzheimer’s, amyotrophic lateral sclerosis (ALS), and schizophrenia.
...07-09-2026, 11:10 AM -
Cancer survival rates have significantly increased in the last few decades in the United States, reaching a combined 70% 5-year survival rate by 2021. Behind this number, there are years of research to find new therapies, drug targets, and early detection methods. But there is one core challenge that keeps slowing down these advances, and it’s about drug resistance.
There is no single reason why many patients don’t respond to treatment as expected. Cancer is...07-08-2026, 05:17 AM
Comment