A comparison between quantitative PCR and droplet digital PCR technologies for circulating microRNA quantification in human lung cancer

BMC Biotechnology, Aug 2016

Background 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. Results 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. Conclusions 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.

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A comparison between quantitative PCR and droplet digital PCR technologies for circulating microRNA quantification in human lung cancer

Campomenosi et al. BMC Biotechnology A comparison between quantitative PCR and droplet digital PCR technologies for circulating microRNA quantification in human lung cancer Paola Campomenosi 0 1 2 Elisabetta Gini 0 2 3 Douglas M. Noonan 2 5 Albino Poli 4 Paola D'Antona 2 3 Nicola Rotolo 3 Lorenzo Dominioni 3 Andrea Imperatori 3 0 Equal contributors 1 The Protein Factory, Centro Interuniversitario di Ricerca in Biotecnologie Proteiche, Politecnico di Milano, ICRM-CNR Milano and University of Insubria , Varese , Italy 2 Department of Biotechnology and Life Sciences (DBSV) and “The Protein Factory”, University of Insubria , Via JH Dunant, 3, 21100 Varese , Italy 3 Department of Surgical Sciences and Human Morphology, DSCM, University of Insubria , Via Guicciardini, 9, 21100 Varese , Italy 4 Department of Public Health and Community Medicine, University of Verona , Verona , Italy 5 Scientific and Technological Pole, IRCCS MultiMedica , Milan , Italy Background: 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. Results: 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. (Continued on next page) - (Continued from previous page) Conclusions: 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. Abbreviations: CV, Coefficient of variation; ddPCR, Droplet digital PCR; miRNA(s), microRNA(s); qPCR, Quantitative real-time PCR Background In recent years efforts have been made to find new approaches for early diagnosis of lung cancer, as the prognosis of this disease strongly correlates with stage at the time of diagnosis. Low-dose computed tomography (CT) screening, the only lung cancer screening method endorsed by leading cancer organizations [ 1–3 ], has uncertain applicability on a population level as a public health measure, due to high cost, undefined cost/benefit ratio, high false-positive rate, overdiagnosis as well as radiation exposure [ 4, 5 ]. Hence, novel methods capable of identifying lung cancer at an early stage are urgently needed. Recently, microRNAs (miRNAs) have been proposed as a promising class of circulating cancer biomarkers: they are very stable in biofluids and the plasma/serum levels of several circulating miRNAs are aberrantly expressed in many diseases, including numerous cancers [ 6, 7 ]. miRNAs are small non-coding RNA molecules of about 20–25 nucleotides that regulate gene expression at the post-transcriptional level [8]. Circulating miRNAs are either stored in particles (exosomes, microvesicles and apoptotic bodies), or associated with RNA-binding proteins or lipoproteins, which prevent their degradation. The abundance and variety of circulating miRNAs suggest a role in cell-cell communication and their potential exploitation as disease biomarkers [ 9–13 ]. Among several miRNAs overexpressed in the serum of patients with lung cancer, miR-21 is the most frequently proposed as biomarker of this disease [ 14–19 ]. Other miRNAs, that appear to function as tumor suppressors, (...truncated)


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Paola Campomenosi, Elisabetta Gini, Douglas Noonan, Albino Poli, Paola D’Antona, Nicola Rotolo, Lorenzo Dominioni, Andrea Imperatori. A comparison between quantitative PCR and droplet digital PCR technologies for circulating microRNA quantification in human lung cancer, BMC Biotechnology, 2016, pp. 60, 16, DOI: 10.1186/s12896-016-0292-7