We have developed an originally designed microfluidic device for single cell isolation [1].For the detection of expressed gene in isolated cells on the device,hot cell-direct RT-PCR method was invented.By the method we can perform cell lysis and RT-PCR in the same reaction chamber without the addition of reagent only by a heat process on the device [2].In this study,we developed an original detection system for expressed gene in isolated single cells in microchamber on the device and succeeded in detection of the RT-PCR product of mRNA in each cells in the microchamber after hot cell-direct RT-PCR.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 a thermal cycler to perform hot cell-direct RT-PCR (Figure 1).By using this detection system,we confirmed the existence of a cell or not in each microchamber on the device prior to hot-cell direct RT-PCR.After cell observation detection of expressed gene in isolated single cells was enabled by performing hot cell-direct RT-PCR on the device in the detection system.In this study,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 at 3500 rpm for 30 sec.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.The fluorescent intensity of microchamber entrapping Jurkat cell increased,but that of the chamber containing no cell did not increase (Figure 2).From relative fluorescent intensity of each chamber,expression of genes was detected by the system.