DYE-FREE DETECTION OF MULTIPLE GENE EXPRESSION BY SEQUENCE-TAGGED MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION COUPLED WITH PYROSEQUENCING FOR DIAGNOSIS OF COLORECTAL CANCER
Early analyzing of the expression of cancer-related genes is an effective means of reducing cancer mortality. Here, we describe a novel sequence-labeling method for quantitatively analyzing different genes expression levels of a sample in a single assay by using Sequence-tagged Multiplex Ligation-dependent Probe Amplification (MLPA) coupled with Pyrosequencing (termed SMAP). Each pair of MLPA probes containing a carefully designed tagged-sequence represents one kind of gene source, thus avoiding the use of a dye for labeling. With a novel tag design on probes, three bases detection for each gene-source is enough for quantifying six genes expression. Moreover the design of each pair of MLPA probes locating in adjacent exons can prevent the DNA contamination in RNA samples. The relative expression levels of six genes, one homekeeping gene (Beta-actin) for controlling and five colorectal cancer (CRC)-related genes (C-myc, CD44v6, Cox-2, H-ras and RAB5A) for diagnosis, have been successfully analyzed based on SMAP. Great differences of the expression levels of CRC-related genes between tumor tissue and normal tissue adjacent to the tumor of a CRC patient were obtained. The sensitivity was high as the cancer diagnosis was based on the total expression levels of all cancer-related genes which were detected simultaneously in SMAP. Therefore, our method has great potential for cancer diagnostics by comprehensive analysis of multiple genes expression.
Gene ezpression sequence tagged MLPA pyrosequencing colorectal cancer
Huang Huan Qi Zongtai Deng Lili Ye Hui Zhou Guohua
Huadong Research Institute for Medicine and Biotechnics, Nanjing 210002, China School of Life Scienc School of Life Science & Technology, China Pharmaceutical University, Nanjing 210009, China Huadong Research Institute for Medicine and Biotechnics, Nanjing 210002, China Medical School, Nanji
国际会议
The 6th International Forum on Post-genome Technologies(6IFPT)(第六届国际后基因组生命科学技术学术论坛)
北京
英文
272-276
2009-09-17（万方平台首次上网日期，不代表论文的发表时间）