title: A comparison between quantitative PCR and droplet digital PCR technologies for circulating microRNA quantification in human lung cancer.
abstract: Selected microRNAs (miRNAs) that are abnormally expressed in the serum of patients with lung cancer have recently been proposed as biomarkers of this disease. The measurement of circulating miRNAs, however, requires a highly reliable quantification method. Quantitative real-time PCR (qPCR) is the most commonly used method, but it lacks reliable endogenous reference miRNAs for normalization of results in biofluids. When used in absolute quantification, it must rely on the use of external calibrators. Droplet digital PCR (ddPCR) is a recently introduced technology that overcomes the normalization issue and may facilitate miRNA measurement. Here we compared the performance of absolute qPCR and ddPCR techniques for quantifying selected miRNAs in the serum.
  
  
  
  In the first experiment, three miRNAs, proposed in the literature as lung cancer biomarkers (miR-21, miR-126 and let-7a), were analyzed in a set of 15 human serum samples. Four independent qPCR and four independent ddPCR amplifications were done on the same samples and used to estimate the precision and correlation of miRNA measurements obtained with the two techniques. The precision of the two methods was evaluated by calculating the Coefficient of Variation (CV) of the four independent measurements obtained with each technique. The CV was similar or smaller in ddPCR than in qPCR for all miRNAs tested, and was significantly smaller for let-7a (p = 0.028). Linear regression analysis of the miRNA values obtained with qPCR and ddPCR showed strong correlation (p < 0.001). To validate the correlation obtained with the two techniques in the first experiment, in a second experiment the same miRNAs were measured in a larger cohort (70 human serum samples) by both qPCR and ddPCR. The correlation of miRNA analyses with the two methods was significant for all three miRNAs. Moreover, in our experiments the ddPCR technique had higher throughput than qPCR, at a similar cost-per-sample.
  
  
  
  Analyses of serum miRNAs performed with qPCR and ddPCR were largely concordant. Both qPCR and ddPCR can reliably be used to quantify circulating miRNAs, however, ddPCR revealed similar or greater precision and higher throughput of analysis.
authors.affiliation: Department of Biotechnology and Life Sciences (DBSV) and "The Protein Factory", University of Insubria, Via JH Dunant, 3, 21100, Varese, Italy. paola.campomenosi@uninsubria.it.|The Protein Factory, Centro Interuniversitario di Ricerca in Biotecnologie Proteiche, Politecnico di Milano, ICRM-CNR Milano and University of Insubria, Varese, Italy. paola.campomenosi@uninsubria.it.
authors.forename: Paola
authors.identifier: 0000-0002-8853-1134
authors.initials: P
authors.lastname: Campomenosi
authors.affiliation: Department of Biotechnology and Life Sciences (DBSV) and "The Protein Factory", University of Insubria, Via JH Dunant, 3, 21100, Varese, Italy.|Department of Surgical Sciences and Human Morphology, DSCM, University of Insubria, Via Guicciardini, 9, 21100, Varese, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Gini
authors.affiliation: Department of Biotechnology and Life Sciences (DBSV) and "The Protein Factory", University of Insubria, Via JH Dunant, 3, 21100, Varese, Italy.|Scientific and Technological Pole, IRCCS MultiMedica, Milan, Italy.
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Noonan
authors.affiliation: Department of Public Health and Community Medicine, University of Verona, Verona, Italy.
authors.forename: Albino
authors.initials: A
authors.lastname: Poli
authors.affiliation: Department of Biotechnology and Life Sciences (DBSV) and "The Protein Factory", University of Insubria, Via JH Dunant, 3, 21100, Varese, Italy.|Department of Surgical Sciences and Human Morphology, DSCM, University of Insubria, Via Guicciardini, 9, 21100, Varese, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: D'Antona
authors.affiliation: Department of Surgical Sciences and Human Morphology, DSCM, University of Insubria, Via Guicciardini, 9, 21100, Varese, Italy.
authors.forename: Nicola
authors.initials: N
authors.lastname: Rotolo
authors.affiliation: Department of Surgical Sciences and Human Morphology, DSCM, University of Insubria, Via Guicciardini, 9, 21100, Varese, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Dominioni
authors.affiliation: Department of Surgical Sciences and Human Morphology, DSCM, University of Insubria, Via Guicciardini, 9, 21100, Varese, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Imperatori
mesh_terms: D014408:Biomarkers, Tumor / Q000097:blood* / Q000235:genetics; D001709:Biotechnology / Q000379:methods; D001774:Blood Chemical Analysis / Q000379:methods; D005591:Chemical Fractionation / Q000379:methods; D005819:Genetic Markers / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood* / Q000175:diagnosis / Q000235:genetics*; D035683:MicroRNAs / Q000097:blood* / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D014408:Biomarkers, Tumor; D005819:Genetic Markers; D035683:MicroRNAs
keywords: Droplet digital PCR; Lung cancer; Serum biomarkers; microRNAs; qPCR