Development of Laser-Mediated Nanodroplet Real-Time PCR on Circulating Tumor Cells (CTC) by Microfilter Platform.

摘要

This project concerns analysis of circulating tumor cells (CTCs), which are proving to be important for prognostics and guiding therapy for breast cancer patients. The project is a collaboration between the University of Miami on the use of microfilters for capture of circulating tumor cells and SRI International on the use of nanodroplet real-time polymerase chain reaction (PCR) for molecular analysis of captured cells. To improve the temperature control for droplet PCR on the University of Miami microfilters, we have extended our DNA melting temperature calibration methodology to use DNA hairpins and dyes that are less sensitive to temperature, adapted our melting and fitting model for the hairpins, have switched the laser heating from a wavelength of 1.45 to 1.9 m, performed sensitivity studies on the influence of water and glycerol on the calibration, and developed software for real-time same-droplet temperature calibration as an image across the droplet. Together with the University of Miami we have produced a strategy for spectrally-multiplexed Taqman to analyze for hMAM, CEA, KRT19 and MUC1 gene expression, including multiplexing optics and relevant primers and probes. We have performed simultaneous multiplexed detection of the four genes using laser-heating PCR in nanoliter droplets at SRI from cDNA prepared at the University of Miami from breast cancer cell lines.