The sequence is 5biotin-AAACACATTCCGGCCCGAG3 and 5biotin-AGCGGTCGATGGTCTTCTG3. between SNC73 expression level and various clinicopathological factors, including sex, age, site, grade of differentiation, depth of invasion and metastases of CRC patients. CONCLUSION: Down-regulation of SNC73 expression may be a relatively specific phenomenon in colorectal cancer. SNC73 is a potential genetic marker for the carcinongenesis of colorectal cancer. The relationship of SNC73 expression and carcinogenesis of colorectal cancer merits further study. == INTRODUCTION == Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in developed western countries[1]. A series of molecular changes are CY3 involved in colorectal carcinogenesis, including activation of oncogenes, inactivation and/or mutational changes of tumor suppressor genes, microsatellite instability, and so on[2-10]. Fearon et al[11] proposed a genetic model of colorectal tumorigenesis. However, despite the tremendous efforts that have been made, there are still many problems unsolved for the model of CRC due to the complexity of carcinogenesis. The early detection and new therapeutic target of CRC have yet to be found. Modern medicine proves that almost all diseases arise from gene function change, which is mainly reflected by the differential gene expression[12]. Hopefully the identification and CY3 characterization of genes expressed differently in tumor tissues and normal mucosa will shed light on the mechanisms of CRC and provide useful molecular markers for screening, diagnosis, prognosis and therapeutic monitoring. To explore new molecular events that are related to carcinogenesis of CRC, Cancer Institute of Zhejiang University constructed CRC negative-associated cDNA libraries by subtractive hybridization[13-17]. Subtractive hybridization between cDNA CY3 of normal mucosal tissues and mRNA of CRC tissues was performed and a total of 46 cDNA clones that were expressed in normal mucosal tissues but were either expressed at a significantly reduced level or not expressed at all in cancerous tissues were isolated. SNC73 is one of the 46 CRC negative-associated complement DNA (cDNA) clones. Northern blot, reverse transcription-polymerase chain reaction (RT-PCR),in situhybridization, andin situPCR confirmed expression of SNC73 in normal epithelial cells and several non-hematopoietic cancer cell strains[17]. The aim of this study was to confirm the negative association between CRC and SNC73 expression and to examine whether such association also exists in other tumors. In the present study, expression level of SNC73 in 90 cases of malignant tumors (31 cases colorectal cancer, 24 cases gastric cancer, 15 cases breast cancer, 11 cases lung cancer and 9 cases liver cancer) and non-cancerous tissues from the same patient was determined by RT-PCR-ELISA. == MATERIALS AND METHODS == == Tissue sample preparation == Fresh samples of surgically resected cancer and its non-cancerous tissues were obtained from the same patient at the Second Affiliated Hospital of Zhejiang University Medical College, and were immediately frozen in liquid nitrogen until CY3 used. Several paired specimens were collected for replication. The total RNA was extracted with Trizol reagent (Gibco BRL, USA). RNA integrity was checked on 1% formaldehyde agarose gel. RNA samples were accepted only when the ratio between absorbance optical density values at 260 nm and at 280 nm was higher than 1.65. == RT-PCR (DIG Labeling) == RNA samples were reverse transcribed with AMV reverse transcriptase (Promega Co.). The primers were labeled with biotin for following immobilization by streptavidin coated microtiter plate modules. The primer for SNC73 was designed based on its cDNA sequence according to previous study. The sequence is 5biotin-AAACACATTCCGGCCCGAG3 and 5biotin-AGCGGTCGATGGTCTTCTG3. The sequence of primer for -actin is 5biotin-TCGACAACGGCTCCGGCA3 and 5biotin-CGTACATGGCTGGGGTGT3. RT-PCR was carried out to amplify the mRNA of SNC73 and -actin. The PCR products were labeled with digoxigenin (dig) by using mixture of dATP, dCTP, dGTP, dTTP and DIG-dUTP in reaction mixture during the amplification RGS7 process. PCR reaction mixture contained 15.7 l sterile water, 2.5 l PCR buffer (10 conc., with MgCl2), 2.5 l 2 mM PCR DIG labeling mix, 2 l 10 mM primers mixture, 0.3 l Taq DNA polymerase and 2 l template cDNA. The cycling program was denaturation of the template 94 C for 3 min, 22 cycles of amplification: 94 C for 10 s (denaturation), 58 C for 20 s (hybridization), 72 C for 30 s (elongation) and elongation (72 C) for 5 min was added after last cycle to ensure the completion of the reaction. PCR products quality was confirmed by electrophoresis.