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

Abstract

BackgroundSelected 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.ResultsIn 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.ConclusionsAnalyses 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0292-7) contains supplementary material, which is available to authorized users.

Isothermal digital detection of microRNA using background-free molecular circuit

“…We calculated the precision of our method to be > 76 % (56 % for RT-qPCR, calculated from the mean value over the three extract concentration tested, Figure 5b). This is in accordance with previous studies showing the improved accuracy of digital PCR in comparison to qPCR (42)(43)(44). As a negative control experiment, the digital quantification of the non-human microRNA mir-39ce using the cognate DNA circuit (presented in Figure 3e), shows the absence of this target in the human extract.…”

“…The quantification by qPCR is estimated from the amplification time (Cq), which is compared to the ones of standard concentrations ( Supplementary Figure 8), assuming the amplification efficiency is the same for both the samples and the standards. However, it has been previously highlighted that imprecision in the PCR calibration or modification of the sample composition significantly affect the quantification (43). This is arguably the origin of the overestimation of the concentration of Let-7a observed here using RT-qPCR.…”

Comparison of droplet digital PCR and direct Sanger sequencing for the detection of the BRAFV600E mutation in papillary thyroid carcinoma

“…After PCR, each droplet either does or does not contain the nucleic acid of interest, allowing estimation of the number of molecules in the reaction under the assumption of a Poisson distribution. The results are expressed as target copies per microliter of the reaction volume …”

“…The results are expressed as target copies per microliter of the reaction volume. 10 The limit of detection of ddPCR is 0.0005%. 11 Therefore, ddPCR analysis sensitively detects and quantifies low-abundance gene mutations like the BRAF V600E mutation.…”

Application of droplet digital PCR in quantitative detection of the cell-free circulating circRNAs

“…(2) It has shown increased sensitivity while detecting low target copies without the need for any pre-amplification steps. (3) It is relatively insensitive to potential PCR inhibitors [20][21][22][23].…”