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

Paola Campomenosi, Elisabetta Gini, Douglas M. Noonan, Albino Poli, Paola D'Antona, Nicola Rotolo, Lorenzo Dominioni, Andrea Imperatori

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Article number60
JournalBMC Biotechnology
Volume16
Issue number1
DOIs
Publication statusPublished - Aug 18 2016

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MicroRNAs
Lung Neoplasms
Technology
Polymerase Chain Reaction
Serum
Tumor Biomarkers
Real-Time Polymerase Chain Reaction
Linear Models
Biomarkers
Regression Analysis

Keywords

  • Droplet digital PCR
  • Lung cancer
  • MicroRNAs
  • QPCR
  • Serum biomarkers

ASJC Scopus subject areas

  • Biotechnology

Cite this

A comparison between quantitative PCR and droplet digital PCR technologies for circulating microRNA quantification in human lung cancer. / Campomenosi, Paola; Gini, Elisabetta; Noonan, Douglas M.; Poli, Albino; D'Antona, Paola; Rotolo, Nicola; Dominioni, Lorenzo; Imperatori, Andrea.

In: BMC Biotechnology, Vol. 16, No. 1, 60, 18.08.2016.

Research output: Contribution to journalArticle

Campomenosi, Paola ; Gini, Elisabetta ; Noonan, Douglas M. ; Poli, Albino ; D'Antona, Paola ; Rotolo, Nicola ; Dominioni, Lorenzo ; Imperatori, Andrea. / A comparison between quantitative PCR and droplet digital PCR technologies for circulating microRNA quantification in human lung cancer. In: BMC Biotechnology. 2016 ; Vol. 16, No. 1.
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AU - Campomenosi, Paola

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AU - Noonan, Douglas M.

AU - Poli, Albino

AU - D'Antona, Paola

AU - Rotolo, Nicola

AU - Dominioni, Lorenzo

AU - Imperatori, Andrea

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N2 - 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.

AB - 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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KW - Lung cancer

KW - MicroRNAs

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KW - Serum biomarkers

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