Abstract

Genomic quantification of isolated single cells on a microfluidic device based on hot cell-direct PCR

Izumi Kubo, Professor, Soka University

? We have developed an original microfluidic device for single cell isolation. For the detection of genes or expressed genes in isolated cells on the device, hot cell-direct PCR/RT-PCR method was invented. By the method we can perform cell lysis and PCR/RT-PCR in the same reaction chamber only by temperature changing of the device. Furthermore, we developed an original detection system for the PCR product in isolated single cells in microchamber on the device. An original detection system was composed of a fluorescent microscope with automatically controllable X-Y stage for observation of isolated single cells in each microchamber and an automatic image acquisition system. Expressed genes in Jurkat human leukemia T cell were examined. At first, Jurkat cells suspended in reaction mixture for hot cell-direct RT-PCR were isolated into the microchambers by the rotation of the device. These isolated Jurkat cells in microchambers could be observed by the detection system. After single cell isolation, hot cell-direct RT-PCR was performed on the device. Hot cell-direct RT-PCR was enabled by using Tth DNA polymerase which has reverse transcriptase activity in the presence of manganese and can be used for RT and PCR. A double-dye fluorescent probe was used to detect the RT-PCR products. The fluorescent signals from RT-PCR products in microchambers were measured by the detection system before and after hot cell-direct RT-PCR. From relative fluorescent intensity of each chamber, expression of genes was detected by the system and it can be applied to the quantification of the genes.