Digital PCR

Absolute quantification of nucleic acids can be determined using digital PCR (dPCR). This method is based on partitioning the sample into many replicate reactions at a limiting dilution resulting in one or zero molecules in each. Digital PCR is an endpoint measurement that provides the ability to quantify nucleic acids without the use of standard curves. This has many advantages over qPCR as it eliminates the need for normalisation controls and is more robust against reagents that may interfere with the efficiency of the PCR reaction (Vogelstein and Kinzler 1999). Droplet digital polymerase chain reaction (ddPCR), the latest technology of dPCR that measures absolute quantities of nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions, was also applied for circulating the miRNAs and the miRNA content of exosomes (Chevillet et al. 2014; Pinheiro et al. 2012). A systematic comparison of ddPCR and qPCR performance for the quantification of miRNAs was performed using synthetic miRNAs as well as sera from 20 patients with advance prostate cancer and 20 age-matched healthy controls (Hindson et al. 2013). The diagnostic sensitivity and specificity of the ddPCR were demonstrated in a platform that used a 20 pl reaction loaded into an eight-channel droplet generation cartridge, each channel dispensed into 20,000 droplets. Discrimination between droplets that did not contain the target (negative) and those that did (positives) was achieved by applying a global fluorescence amplitude threshold. The two methods displayed comparable sensitivity, but ddPCR had greater precision and higher reproducibility than qPCR.

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