A second-generation profiling system for quantitative methylation analysis of multiple gene promoters: application to lung cancer.

Cancer-specific gene promoter methylation has been described in many types of cancers, and various semi-quantified results have shown their usefulness. Here, we show a more sensitive and specific second-generation system for profiling the DNA methylation status. This method is based on bisulfite reaction of DNA and real-time PCR using two TaqMan MGB probes labeled with different fluorescence, followed by clustering analysis. Primers were designed with CpG-less sequences, and TaqMan MGB probes were designed to contain three or four CpG sites and to be shorter than conventional TaqMan probes. We have added new criteria for primer and probe design for further specificity. We confirmed the reliability of this system and applied it to analysis of lung cancers. Using 10 promoters, 90 primary lung cancers were clustered into six groups consisting of cases having similar smoking status and pathological findings. EGFR mutation and p16 promoter DNA methylation were exclusive, as previously reported; however, DNA methylation in other genes was unrelated to EGFR mutation. This system was also useful to distinguish double primary lung cancers from a single cancer with intrapulmonary metastasis. As above, our system has widespread availability in clinical use and biological research.

Data processing for multi-channel optical recording: action potential detection by neural network.

Using a neural network, we have developed a program for fast and precise detection of action potentials (AP) in raw multi-channel optical recording data. The AP detection was performed in two steps: first, peaks were detected in raw optical data, and, second, the peaks were classified by the neural network into APs, noise and undecided peaks. The network was optimized and trained by the backpropagation learning algorithm, employing some thousands of manually classified peaks. The performance of the optimized network was found to be not completely satisfactory, although it was better than the classification by template matching and nearest-neighbor rules. The addition of a signal-to-noise ratio (SNR) of a peak to the network classification improved the classification performance: in comparison with the manual classification results, 96% of manually classified APs were detected. The causes of classification errors were discussed. In spite of the fact that the program required a slight amount of human intervention for undecided peaks, the program could allow mostly automatic AP detection.

A precision method to measure average viscoelastic parameters of erythrocyte populations.

Suspensions of erythrocytes in media of low conductivity are subjected to homogenous high frequency electric fields (1 MHz, approximately 10 to 40 kV/m). The resulting transient deformation of the cells is measured by laser light diffraction. Employing a viscoelastic model of the erythrocyte membrane, relative values of membrane shear modulus and response time can currently be determined to within 7% or better. With a measurement time of one minute the average values of some 10(5) cells can be obtained. As a test of the method, osmotic swelling and deflation of the cells and crosslinking their membrane skeleton by diamide are used to alter the viscoelastic properties of the erythrocytes.