Isolation of nucleic acids from low biomass samples: detection and removal of sRNA contaminants

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.Due to its inherent instability, contamination with RNA is usually considered to be unlikely. 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 qPCRbased 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 'ultra-clean' extraction kits was confirmed. In 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.

MicroRNAs in bovine milk exosomes are bioavailable in humans but do not elicit a robust pro-inflammatory cytokine response

“…A previous report suggested that miR analysis may be confounded by contamination of NucleoSpin miR plasma columns with microbial RNAs, but we could not reproduce these findings [30]. We tested for column contamination by passing molecular biology grade water through hypochlorite-treated and non-treated columns and compared the Ct values of the six miRs in the two treatments by RT-qPCR (n = 5 per treatment).…”

“…MiRs were isolated from plasma by using the NucleoSpin miRNA plasma kit (Macherey-Nagel) and assayed by reverse transcription quantitative PCR (RT-qPCR) as described above. Heintz-Buschart et al reported that spin columns for RNA purification may be contaminated with microbial RNAs and produce false positive results in miR analysis [30]. Although we could not reproduce contamination of spin columns, we opted to err on the side of caution and purified columns with 0.5% sodium hypochlorite prior to use [30].…”

“…Ct values were greater than 35 in all samples tested. Absence of contamination notwithstanding, we erred on the side of caution and treated columns with 0.5% sodium hypochlorite prior to miR extraction from plasma [30].…”

“…Heintz-Buschart et al reported that spin columns for RNA purification may be contaminated with microbial RNAs and produce false positive results in miR analysis [30]. Although we could not reproduce contamination of spin columns, we opted to err on the side of caution and purified columns with 0.5% sodium hypochlorite prior to use [30]. The areas under the curves (AUCs) of plasma miRs concentration vs time curves were calculated by using the linear trapezoidal rule, and were used to assess the apparent bioavailability of miRs [55].…”

The Genetics, Biochemistry, and Biophysics of Carbon Cycling by Deep Life

“…Recent work has highlighted that commercial DNA/RNA extraction kit reagents, typically designed for high-biomass environments, contain their own microbiome that can skew downstream amplicon data analyses (46,47). It is also important to remove reagent contamination from SAGs, metagenomes, and metatranscriptomes (48)(49)(50). Thus, great care must be taken to screen resulting reads and assembled contigs to identify and remove suspicious sequences, while also allowing that sometimes organisms that were previously thought to represent surface contamination, such as cyanobacteria, may actually be legitimate community members (51).…”