Multicenter Evaluation of Circulating Plasma MicroRNA Extraction Technologies for the Development of Clinically Feasible Reverse Transcription Quantitative PCR and Next-Generation Sequencing Analytical Work Flows
Jazyk angličtina Země Anglie, Velká Británie Médium print-electronic
Typ dokumentu časopisecké články, multicentrická studie, práce podpořená grantem
PubMed
31235535
DOI
10.1373/clinchem.2019.303271
PII: clinchem.2019.303271
Knihovny.cz E-zdroje
- MeSH
- Caenorhabditis elegans chemie MeSH
- chemická frakcionace metody MeSH
- cirkulující mikroRNA krev izolace a purifikace MeSH
- extracelulární vezikuly chemie MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- lidé středního věku MeSH
- lidé MeSH
- nádorové biomarkery krev izolace a purifikace MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- senioři MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- zvířata MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cirkulující mikroRNA MeSH
- nádorové biomarkery MeSH
BACKGROUND: In human body fluids, microRNA (miRNA) can be found as circulating cell-free miRNA (cfmiRNA), as well as secreted into extracellular vesicles (EVmiRNA). miRNAs are being intensively evaluated as minimally invasive liquid biopsy biomarkers in patients with cancer. The growing interest in developing clinical assays for circulating miRNA necessitates careful consideration of confounding effects of preanalytical and analytical parameters. METHODS: By using reverse transcription quantitative real-time PCR and next-generation sequencing (NGS), we compared extraction efficiencies of 5 different protocols for cfmiRNA and 2 protocols for EVmiRNA isolation in a multicentric manner. The efficiency of the different extraction methods was evaluated by measuring exogenously spiked cel-miR-39 and 6 targeted miRNAs in plasma from 20 healthy individuals. RESULTS: There were significant differences between the tested methods. Although column-based extraction methods were highly effective for the isolation of endogenous miRNA, phenol extraction combined with column-based miRNA purification and ultracentrifugation resulted in lower quality and quantity of isolated miRNA. Among all extraction methods, the ubiquitously expressed miR-16 was represented with high abundance when compared with other targeted miRNAs. In addition, the use of miR-16 as an endogenous control for normalization of quantification cycle values resulted in a decreased variability of column-based cfmiRNA extraction methods. Cluster analysis of normalized NGS counts clearly indicated a method-dependent bias. CONCLUSIONS: The choice of plasma miRNA extraction methods affects the selection of potential miRNA marker candidates and mechanistic interpretation of results, which should be done with caution, particularly across studies using different protocols.
Bayer AG Pharmaceutical Division Biomarker Research Wuppertal Germany
Bayer AG Pharmaceutical Division Biomarker Research Wuppertal Germany;
Bayer AG Pharmaceutical Division Translational Assay Technology Berlin Germany
Institute for Molecular Medicine Finland University of Helsinki Helsinki Finland
Institute of Biotechnology CAS v v i Vestec Czech Republic
Integrated BioBank of Luxembourg Dudelange Luxembourg
Orion Pharma Orion Corporation Espoo Finland
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