title: Optimization and Standardization of Circulating MicroRNA Detection for Clinical Application: The miR-Test Case.
abstract: The identification of circulating microRNAs (miRNAs) in the blood has been recently exploited for the development of minimally invasive tests for the early detection of cancer. Nevertheless, the clinical transferability of such tests is uncertain due to still-insufficient standardization and optimization of methods to detect circulating miRNAs in the clinical setting.
  
  
  
  We performed a series of tests to optimize the quantification of serum miRNAs that compose the miR-Test, a signature for lung cancer early detection, and systematically analyzed variables that could affect the performance of the test. We took advantage of a large-scale (>1000 samples) validation study of the miR-Test that we recently published, to evaluate, in clinical samples, the effects of analytical and preanalytical variables on the quantification of circulating miRNAs and the clinical output of the signature (risk score).
  
  
  
  We developed a streamlined and standardized pipeline for the processing of clinical serum samples that allows the isolation and analysis of circulating miRNAs by quantitative reverse-transcription PCR, with a throughput compatible with screening trials. The major source of analytical variation came from RNA isolation from serum, which could be corrected by use of external (spike-in) or endogenous miRNAs as a reference for normalization. We also introduced standard operating procedures and QC steps to check for unspecific fluctuations that arise from the lack of standardized criteria in the collection or handling of the samples (preanalytical factors).
  
  
  
  We propose our methodology as a reference for the development of clinical-grade blood tests on the basis of miRNA detection.
authors.affiliation: Center for Genomic Science of IIT@SEMM, Istituto Italiano di Tecnologia, Milan, Italy;
authors.forename: Matteo Jacopo
authors.initials: MJ
authors.lastname: Marzi
authors.affiliation: Molecular Medicine Program, European Institute of Oncology, Milan, Italy;
authors.forename: Francesca
authors.initials: F
authors.lastname: Montani
authors.affiliation: Molecular Medicine Program, European Institute of Oncology, Milan, Italy; The FIRC Institute for Molecular Oncology Foundation (IFOM), Milan, Italy;
authors.forename: Rose Mary
authors.initials: RM
authors.lastname: Carletti
authors.affiliation: Molecular Medicine Program, European Institute of Oncology, Milan, Italy;
authors.forename: Fabio
authors.initials: F
authors.lastname: Dezi
authors.affiliation: Molecular Medicine Program, European Institute of Oncology, Milan, Italy; Division of Epidemiology and Biostatistics.
authors.forename: Elisa
authors.initials: E
authors.lastname: Dama
authors.affiliation: Molecular Medicine Program, European Institute of Oncology, Milan, Italy;
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Bonizzi
authors.affiliation: Division of Laboratory Medicine.
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Sandri
authors.affiliation: Division of Radiology, and.
authors.forename: Cristiano
authors.initials: C
authors.lastname: Rampinelli
authors.affiliation: Division of Radiology, and.
authors.forename: Massimo
authors.initials: M
authors.lastname: Bellomi
authors.affiliation: Division of Epidemiology and Biostatistics.
authors.forename: Patrick
authors.initials: P
authors.lastname: Maisonneuve
authors.affiliation: Division of Thoracic Surgery, European Institute of Oncology, Milan, Italy;
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Spaggiari
authors.affiliation: Division of Thoracic Surgery, European Institute of Oncology, Milan, Italy; Humanitas Research Hospital, Thoracic Surgery, Rozzano, Milan, Italy;
authors.forename: Giulia
authors.initials: G
authors.lastname: Veronesi
authors.affiliation: Molecular Medicine Program, European Institute of Oncology, Milan, Italy;
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Bianchi
authors.affiliation: Molecular Medicine Program, European Institute of Oncology, Milan, Italy; The FIRC Institute for Molecular Oncology Foundation (IFOM), Milan, Italy; Dipartimento di Scienze della Salute, University of Milan, Milan, Italy. francesco.nicassio@iit.it pierpaolo.difiore@ieo.eu.
authors.forename: Pier Paolo
authors.initials: PP
authors.lastname: Di Fiore
authors.affiliation: Center for Genomic Science of IIT@SEMM, Istituto Italiano di Tecnologia, Milan, Italy; Molecular Medicine Program, European Institute of Oncology, Milan, Italy; francesco.nicassio@iit.it pierpaolo.difiore@ieo.eu.
authors.forename: Francesco
authors.initials: F
authors.lastname: Nicassio
mesh_terms: D006801:Humans; D035683:MicroRNAs / Q000097:blood* / Q000235:genetics* / Q000592:standards; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*
chemical_list: D035683:MicroRNAs