Hi all, I submitted 35 bacterial total RNA samples from lab bioaerosol experiments for mRNA-seq yesterday. The sequencing partner wrote this back after basic QC/QA:
All of the samples have a very prominent band at 60bp. Most RNA preps will have small RNA bands but it is usually a small fraction of the whole sample. Your 60bp band is the majority of the RNA in a lot of these samples and still very strong in all the rest. It actually looks as if you have enriched for small RNAs somehow. This 60bp band doesn’t appear to be from degradation, because the Ribosomal peak ratios indicate that it’s in pretty good shape, it just has a very odd profile otherwise.
Has anyone ever seen anything like this before? 60bp throughout is....confusing.
The samples are mostly of a Mycobacterium spp. and a few of P. aeruginosa. The processing was (1) bioaerosol collection into RNAprotect solution, (2) extraction with lysozyme, proteinaseK, and a Zymo Quick-RNA kit, and (3) quantification on qubit / tapestation.
The best explanations I can imagine are the RNAprotect enriched somehow for small RNAs, or there's incomplete lysis of the difficult-to-crack Mycobacterial membrane (which I originally optimized for), or there's some contamination of some sort in my enzymes. None of these explanations feel quite right but I'm at a loss for what could've happened here.
Any and all insight appreciated!
Emily Kraus A difficult mystery! I assume that both the mycobacterial and pseudomonas samples have the 60 bp RNA--that seems to be what the note from your sequencing partner indicates. If so, then it could not be due to some biological characteristic specific to one these types. The only alternative that I can think of is that it was introduced as part of the RNA extraction procedure that was done with all of the samples. (Was some "carrier" introduced at some extraction step?) I would go carefully over each step and each reagent used (and possibly the recent history of those reagents) to try to figure out where and how such a large amount of contaminant might have been introduced. If you did any adjustments to the RNA preps to standardize the amounts that you sent to your sequence partner or used different amounts of a particular reagent on some samples, you might look to see if those differences correspond to the variation in the amount of 60 bp in the samples. If it is present in massive amounts, the contaminant might also be detected just by checking the RNA content of a reagent/resuspension solution.
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