Small RNA profiling of low biomass samples: identification and removal of contaminants
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12915_2018_Article_522.pdf
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Author(s)
Heintz-Buschart, Anna
Yusuf, Dilmurat
Kaysen, Anne
Etheridge, Alton
Fritz, Joëlle V
May, Patrick
de Beaufort, Carine
Upadhyaya, Bimal B
Galas, David J
Wilmes, Paul
Date Issued
May 2018
Journal
BMC Biology
Publisher
BioMed Central Ltd
Citation
Heintz-Buschart, Anna et al. "Small RNA profiling of low biomass samples: identification and removal of contaminants." BMC Biology 16 (May 2018): 52
Version
Final published version
Abstract
Background
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. 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.
Results
Herein, 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.
Conclusion
This 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. Keywords: RNA sequencing; Artefact removal; Exogenous RNA in human blood plasma; Contaminant RNA; Spin columns
MIT Department
Massachusetts Institute of Technology. Department of Biology
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Creative Commons Attribution
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DOI of Published Version
https://doi.org/10.1186/s12915-018-0522-7
