Τίτλος:
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
Γλώσσες Τεκμηρίου:
Αγγλικά
Περίληψη:
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. © 2019 American Association for Clinical Chemistry.
Συγγραφείς:
Kloten, V.
Neumann, M.H.D.
Pasquale, F.D.
Sprenger-Haussels, M.
Shaffer, J.M.
Schlumpberger, M.
Herdean, A.
Betsou, F.
Ammerlaan, W.
af Hällström, T.
Serkkola, E.
Forsman, T.
Lianidou, E.
Sjöback, R.
Kubista, M.
Bender, S.
Lampignano, R.
Krahn, T.
Schlange, T.
for the CANCER-ID consortium
Περιοδικό:
Advances in Clinical Chemistry
Εκδότης:
American Association for Clinical Chemistry Inc.
Λέξεις-κλειδιά:
microRNA; microRNA 122; microRNA 150; microRNA 16; microRNA 191; microRNA 21; unclassified drug; circulating microRNA; tumor marker, adult; Article; blood donor; blood sampling; cluster analysis; controlled study; exosome; female; human; liquid biopsy; male; middle aged; multicenter study; next generation sequencing; normal human; real time polymerase chain reaction; real time reverse transcription polymerase chain reaction; reverse transcription; RNA extraction; ultracentrifugation; aged; animal; blood; Caenorhabditis elegans; chemistry; clinical trial; fractionation; high throughput sequencing; isolation and purification; procedures; reverse transcription polymerase chain reaction, Aged; Animals; Biomarkers, Tumor; Caenorhabditis elegans; Chemical Fractionation; Circulating MicroRNA; Extracellular Vesicles; Female; High-Throughput Nucleotide Sequencing; Humans; Male; Middle Aged; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction
DOI:
10.1373/clinchem.2019.303271
