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

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.

Keywords: Droplet digital PCR; Lung cancer; Serum biomarkers; microRNAs; qPCR.

Fig. 1

Workflow of the experiments. a In the first experiment, for determination of each miRNA of interest (miR-21, miR-126 and let-7a) in each of 15 serum samples, we performed 4 independent qPCRs and 4 independent ddPCRs. b In the second experiment, the miRNAs of interest were measured with qPCR and ddPCR in 70 serum samples. All qPCRs were run in triplicate; ddPCRs were run as single reactions

Fig. 2

ddPCR frequently shows greater precision compared to qPCR for quantifying miRNAs under study. Scatter plots showing the expression values of the selected miRNAs, by qPCR (panels on the left) and ddPCR (panels on the right), in the 15 samples (first experiment). Four independent amplifications were performed for each sample in each technique. For qPCR each dot represents the mean of the technical triplicates. The mean and the standard deviation of values obtained from the four independent measurements are shown for each of the 15 samples

Fig. 3

Correlation between qPCR and ddPCR measurements in the first experiment. miR-21, miR-126 and let-7a levels (copies/microliter) were measured in 15 serum samples. Each dot represents the average of 4 independent determinations

Fig. 4

Correlation between qPCR and ddPCR measurements in the second experiment. miR-21, miR-126 and let-7a levels (copies/microliter) were measured in 70 serum samples