Abstract
BackgroundSequencing-based analyses of low-biomass samples are known to be prone to misinterpretation due to the potential presence of contaminating molecules derived from laboratory reagents and environments. DNA contamination has been previously reported, yet contamination with RNA is usually considered to be very unlikely due to its inherent instability. Small RNAs (sRNAs) identified in tissues and bodily fluids, such as blood plasma, have implications for physiology and pathology, and therefore the potential to act as disease biomarkers. Thus, the possibility for RNA contaminants demands careful evaluation.ResultsHerein, we report on the presence of small RNA (sRNA) contaminants in widely used microRNA extraction kits and propose an approach for their depletion. We sequenced sRNAs extracted from human plasma samples and detected important levels of non-human (exogenous) sequences whose source could be traced to the microRNA extraction columns through a careful qPCR-based analysis of several laboratory reagents. Furthermore, we also detected the presence of artefactual sequences related to these contaminants in a range of published datasets, thereby arguing in particular for a re-evaluation of reports suggesting the presence of exogenous RNAs of microbial and dietary origin in blood plasma. To avoid artefacts in future experiments, we also devise several protocols for the removal of contaminant RNAs, define minimal amounts of starting material for artefact-free analyses, and confirm the reduction of contaminant levels for identification of bona fide sequences using ‘ultra-clean’ extraction kits.ConclusionThis is the first report on the presence of RNA molecules as contaminants in RNA extraction kits. The described protocols should be applied in the future to avoid confounding sRNA studies.
Highlights
Sequencing-based analyses of low-biomass samples are known to be prone to misinterpretation due to the potential presence of contaminating molecules derived from laboratory reagents and environments
To rule out sequencing errors or contamination during sequencing library preparation, a real-time quantitative polymerase chain reaction (qPCR) assay was developed to assess the presence of non-human sequences in the small RNA (sRNA) preparations from plasma
QPCR assays for putative exogenous sRNAs in human blood plasma Synthetic sRNAs with the putative exogenous sequences found in plasma were poly-adenylated and reverse transcribed to yield cDNA, and used for optimisation of PCR primers and conditions (Table 1)
Summary
Sequencing-based analyses of low-biomass samples are known to be prone to misinterpretation due to the potential presence of contaminating molecules derived from laboratory reagents and environments. Small RNAs (sRNAs) identified in tissues and bodily fluids, such as blood plasma, have implications for physiology and pathology, and the potential to act as disease biomarkers. Diet-derived exogenous microRNAs have been proposed to exert an influence on human physiology [31, 32], but these findings have been refuted by others due to a lack of reproducibility in validation studies [33,34,35,36,37] This discussion happens at a time when DNA sequencing-based analyses of low-biomass samples have been recognised as prone to being confounded by contaminants [38]. From initial sample handling [39], to extraction kits [40], to sequencing reagents [41], multiple sources of DNA contamination and artefactual sequencing data have been described
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