TY - JOUR
T1 - 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
AU - Kloten, Vera
AU - Neumann, Martin H.D.
AU - Pasquale, Francesca Di
AU - Sprenger-Haussels, Markus
AU - Shaffer, Jonathan M.
AU - Schlumpberger, Martin
AU - Herdean, Andrei
AU - Betsou, Fay
AU - Ammerlaan, Wim
AU - af Hällström, Taija
AU - Serkkola, Elina
AU - Forsman, Tarja
AU - Lianidou, Evi
AU - Sjöback, Robert
AU - Kubista, Mikael
AU - Bender, Sebastian
AU - Lampignano, Rita
AU - Krahn, Thomas
AU - Schlange, Thomas
AU - for the CANCER-ID consortium
N1 - Funding Information:
Research Funding: The authors participate in the Innovative Medicines Initiative consortium CANCER-ID. CANCER-ID is supported by the Innovative Medicines Initiative (IMI) Joint Undertaking under grant agreement n°115,749, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007–2013) and EFPIA companies’ in-kind contributions. RVO: 86652036 and BIOCEV (CZ.1.05/1.1.00/02.0109) from the ERD to institution. M. Kubista, Grant Agency of the Czech Republic 17-24441S and 17-04034S. Expert Testimony: None declared. Patents: M. Schlumpberger, PCT/EP2017/074490.
Publisher Copyright:
© 2019 American Association for Clinical Chemistry.
PY - 2019
Y1 - 2019
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85071784993&partnerID=8YFLogxK
U2 - 10.1373/clinchem.2019.303271
DO - 10.1373/clinchem.2019.303271
M3 - Article
C2 - 31235535
AN - SCOPUS:85071784993
SN - 0009-9147
VL - 65
SP - 1132
EP - 1140
JO - Clinical Chemistry
JF - Clinical Chemistry
IS - 9
ER -