mRNA contamination in miRNA prep

Question

[Abhishek]

Hi all,

I'm performing downstream analysis for biomarker discovery on samples obtained with QIAseq miRNA Library prep and NovaSeq sequencing. I used Qiagen Geneglobe for quantification and obtained 460 miRNA + piRNA and 825 mRNA. We were not expecting to see mRNAs, and that too in this large proportion.

Is there some specific QC I could perform to check this ? (I only have the fastq files from sequencing)

The sequencing centre suggested that the samples provided might be degraded, and asked if I have obtained RIN for the total RNA. (The sequencing centre was sent only the small RNA and I only have the fastq files).

Is it possible to obtain RIN values from fastq files ? From what I understand (from https://bmcmolbiol.biomedcentral.com/articles/10.1186/1471-2199-7-3), RIN should be obtained during sample prep and not at later stages ?

Answer 1

[Trivas]

You're correct. You would have to run a separate assay (TapeStation) using your total RNA and not your small RNA prep to calculate a RIN score. I'm not too familiar with Geneglobe, but I would look into a second method of quantifying the amount of mRNA in your sequencing runs.

[Abhishek]

Thank you Trivas. Is there some specific QC (other than RIN) I could perform on the fastq files (or quantified result), to understand the reason for large proportion of mRNA ?