Absolute quantification of lung cancer related microRNA by droplet digital PCR

Digital polymerase chain reaction (digital PCR) enables the absolute quantification of nucleic acids through the counting of single molecules, thus eliminating the need for standard curves or endogenous controls. In this study, We developed a droplet digital PCR (ddPCR) system based on an oil saturated PDMS (OSP) microfluidic chip platform for quantification of lung cancer related microRNA (miRNA). The OSP chip was made with PDMS and was oil saturated to constrain oil swallow and maintain the stability of droplets. Two inlets were designed for oil and sample injection with a syringe pump at the outlet. Highly uniform monodisperse water-in-oil emulsion droplets to be used for subsequent detection and analysis were generated at the cross section of the channel. We compared miRNA quantification by the ddPCR system and quantitative real-time PCR (qPCR) to demonstrate that the ddPCR system was superior to qPCR both in its detection limit and smaller fold changes measurement. This droplet PCR system provides new possibilities for highly sensitive and efficient detection of cancer-related genes. (C) 2015 Elsevier B.V. All rights reserved.