Abstract
BackgroundCirculating microRNAs (miRNAs) are attractive non-invasive biomarkers for a variety of conditions due to their stability and altered pathophysiological expression levels. Reliable detection of global expression profiles is required to maximise miRNA biomarker discovery. Although developments in small RNA-Seq technology have improved detection of plasma-based miRNAs, the low RNA content and sequencing bias introduced during library preparation remain challenging. In this study we compare commercially available RNA extraction methods using MagnaZol (Bioo Scientific) or miRNeasy (QIAGEN) and three library preparation methods - CleanTag (TriLink), NEXTflex (Bioo Scientific) and QIAseq (QIAGEN) - which aim to address one or both of these issues.ResultsDifferent RNA extractions and library preparation protocols result in differential detection of miRNAs. A greater proportion of reads mapped to miRNAs in libraries prepared with MagnaZol RNA than with miRNeasy RNA. Libraries prepared using QIAseq demonstrated the greatest miRNA diversity with many more very low abundance miRNAs detected (~ 2–3 fold more with < 10 reads), whilst CleanTag detected the fewest individual miRNAs and considerably over-represented miR-486-5p. Libraries prepared with QIAseq had the strongest correlation with RT-qPCR quantification. Analysis of unique molecular indices (UMIs) incorporated in the QIAseq protocol indicate that little PCR bias is introduced during small RNA library preparation.ConclusionsSmall RNAs were consistently detected using all RNA extraction and library preparation protocols tested, but with some miRNAs at significantly different levels. Choice of the most suitable protocol should be informed by the relative importance of minimising the total sequencing required, detection of rare miRNAs or absolute quantification.
Highlights
Circulating microRNAs are attractive non-invasive biomarkers for a variety of conditions due to their stability and altered pathophysiological expression levels
Wider adoption of Next generation sequencing (NGS) to detect miRNAs and other small RNAs is hampered by biases which mean that the expression values measured for miRNAs
Our results show that whilst all protocols provided reproducible results, which can be used for comparison of relative expression, the miRNA profile detected from plasma samples is greatly affected by the choice of library preparation kit and, to a lesser extent, the RNA extraction method
Summary
Circulating microRNAs (miRNAs) are attractive non-invasive biomarkers for a variety of conditions due to their stability and altered pathophysiological expression levels. Developments in small RNA-Seq technology have improved detection of plasma-based miRNAs, the low RNA content and sequencing bias introduced during library preparation remain challenging. Wider adoption of NGS to detect miRNAs and other small RNAs (small RNA-Seq) is hampered by biases which mean that the expression values measured for miRNAs. Wong et al BMC Genomics (2019) 20:446 may not accurately reflect their absolute levels. It has been shown that ligation bias is determined by preferential secondary structures formed between miRNAs and adapters and that it can be reduced by use of adapters with degenerate bases [8,9,10,11]
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