DIGITAL DETECTION OF MULTIPLE GENES EXPRESSED IN COLORECTAL CANCER BY COUNTING BEADS COATED WITH EMULSION PCR PRODUCTS

摘要

The early detection of cancer through analysis of gene expression could have a substantial impact on morbidity and mortality. For this purpose, it is essential to determine the low levels of the abnormal genes expression that are considered as biomarkers of malignancy during the initiation of cancer. To achieve this goal, we have proposed a novel method that is sensitive enough to detect the gene expression level three orders of magnitude lower than that of house-keeping gene (beta-actin) by using amplicon-coated microbeads and multiple single-molecule-PCR in water-in-oil emulsions coupled with the hydrogel bead-chip. In our method, cDNA sample are directly used as the starting template for the multiple emulsion PCR amplification by several gene-specific primers. The recovered beads reflecting the diversity of cDNA sample are immobilized through hydrogel and digitally counted. The quantitative performance is good as the correlation between the measured and theoretical ratios of the two colored beads reached 0. 999. Moreover, tumor cell (SW480), tumor tissue and normal tissue adjacent to the tumor of the colorectal cancer (CRC) patients were analyzed by our method. Great differences of the expression levels of CRC-related genes between tumor tissue and normal tissue were obtained, suggesting that our method is promising for early detection of cancer.