Small RNA profiling of low biomass samples: identification and removal of contaminants

BMC Biology
Anna Heintz-BuschartPaul Wilmes

Abstract

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. 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 RN...Continue Reading

Associated Software

Mar 29, 2018·Patrick May, Anna Heintz-Buschart

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Citations

Oct 5, 2018·Annual Review of Animal Biosciences·Janos ZempleniEzra Mutai
Jun 7, 2019·Journal of Pediatric Gastroenterology and Nutrition·Amy LeifermanJanos Zempleni
Feb 20, 2020·RNA Biology·Juan Pablo Tosar, Alfonso Cayota
Aug 22, 2018·Journal of Extracellular Vesicles·Ryan M AllenKasey C Vickers
Nov 27, 2018·RNA Biology· SanchitaPrabodh Kumar Trivedi
Jan 9, 2020·Cell & Bioscience·Bernardetta LeddaMariangela Miele
Sep 23, 2020·Annals of the Rheumatic Diseases·Michelle J OrmsethC Michael Stein
Apr 9, 2021·Forensic Science, Medicine, and Pathology·Sarah IshakJennifer M Young
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Jun 10, 2021·BMC Genomics·Geneviève BartSeppo J Vainio
Jul 2, 2021·Frontiers in Genetics·Susana Martínez ArbasShaman Narayanasamy

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Datasets Mentioned

BETA
PRJNA419919
AE006468

Methods Mentioned

BETA
PCR
chip

Software Mentioned

- toolkit
CD
R
megablast
- EST
Novoalign
FASTX
- 2D
FastQC
blastn

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