RT Journal Article
SR Electronic
T1 Isolation of nucleic acids from low biomass samples: detection and removal of sRNA contaminants
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 232975
DO 10.1101/232975
A1 Heintz-Buschart, Anna
A1 Yusuf, Dilmurat
A1 Kaysen, Anne
A1 Etheridge, Alton
A1 Fritz, Joëlle V.
A1 May, Patrick
A1 de Beaufort, Carine
A1 Upadhyaya, Bimal B.
A1 Ghosal, Anubrata
A1 Galas, David J.
A1 Wilmes, Paul
YR 2018
UL http://biorxiv.org/content/early/2018/01/03/232975.abstract
AB 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. Due to its inherent instability, contamination with RNA is usually considered to be unlikely.Results Here we report the presence of small RNA (sRNA) contaminants in widely used microRNA extraction kits and means for their depletion. Sequencing of sRNAs extracted from human plasma samples was performed and significant levels of non-human (exogenous) sequences were detected. The source of the most abundant of these sequences could be traced to the microRNA extraction columns by qPCR-based analysis of laboratory reagents. The presence of artefactual sequences originating from the confirmed contaminants were furthermore replicated in a range of published datasets. To avoid artefacts in future experiments, several protocols for the removal of the contaminants were elaborated, minimal amounts of starting material for artefact-free analyses were defined, and the reduction of contaminant levels for identification of bona fide sequences using ‘ultraclean’ extraction kits was confirmed.Conclusion This is the first report of the presence of RNA molecules as contaminants in laboratory reagents. The described protocols should be applied in the future to avoid confounding sRNA studies.LIST OF ABBREVIATIONSqPRC: real-time quantitative polymerase chain reactionsRNA: small RNA