Construction And Selection Of Subtracted CDNA Library Of Mouse Hepatocarcinoma Cell Lines With Different Lymphatic Metastasis Potential

Background: Recently, studies on mechanism of tumor metastasis have stepped into a stage of multiple genes age. However, metastasis related genes available nowadays show little capability to elucidate the puzzling process of metastasis, therefore, more attention are paid to screen candidate genes of metastasis high throughput. There are many different techique to detect the difference of gene expression between the cells with different biological phenotypes, such as Expressed sequencing tag(EST), Serial analysis of gene expression(SAGE), subtractive hybridization, mRNA differential display (DD-RTPCR), cDNA representative difference analysis (RDA), suppressive subtractive hybridization(SSH), and cDNA microarry. As one of the high throughput screening technique, SSH technique is characteristic by two distinct advantages: (1) it boasts a high subtraction efficiency; (2) it harbors an equalized representation of differentially expressed sequences which can separate effectively both high and low copy expressed genes mainly because of normalization. Hca-F and Hca-P are a pair of synogenetic mouse hepatocarcinoma ascites cell lines, presenting a specific potential of lymphogenetic metastasis when inoculated subcutaneously in 615 mice, Hca-F showing a high metastatic potential(>80%), while Hca-P a low one(<30%) Synogenetic and specific for lymphogenetic metastasis, with significant differentiation only in metastatic potential, candidate genes involved in lymphogenetic metastasis are supported to be obtained between them.Object: To make the molecular mechanism of lymphatic metastasis clearly, We employ suppressive subtracted hybridazation(SSH) technique to identify differentially expressed genes special for Hca-P so as to obtain candidate suppressive genes of lymphogenetic metastasis, we try to detect the difference of gene expression betweenmouse hepatocarcinoma cell lines with different lymphatic metastasis potential using SSH method.Method: Isolation of total RNA was performed by TRIZOLTM(GIBCOBRL) and mRNA was carried out according to the protocol of oligotex mRNA spin column purification kit (Qiagen). The quantity and integrity of mRNA were detected by ultraviolet spectrometer and by electrophoresing on a denaturing formaldehyde agarose stained by EtBr. mRNA of Hca-P serves as tester and mRNA of Hca-F as driver. SSH was performed between tester and driver using a PCR select?cDNA subtraction kit and 50PCR enzyme kit (Clontech, Heidelberg, Germany) following the instructions of the manufacturer. Briefly, 2g aliquots each of poly (A+) mRNA from the tester and the pooled driver were subjected to cDNA synthesis. Thereafter, they were purified by passing through Chroma spin-400 columns (Clontech, USA). Each purified tester cDNA was digested with Rsa I . The tester cDNAs were subdivided into two equal groups and then ligated to adaptor 1 and 2R in separate ligation reactions, insure ligation efficiency >25%.Subtractive hybridization was performed by annealing an excess of driver cDNAs with each sample of adaptor-ligated tester cDNAs. The cDNAs were heat-denatured and incubated in 68 for 8h. After the first hybridization, the two samples were mixed together and hybridized again with freshly heat-denatured driver cDNAs for 20h at 68. Two rounds of hybridization would generate a normalized population of tester-specific cDNAs with different adaptors on each end. After filling in the ends, two rounds of PCR amplification were performed to enrich desired cDNAs containing both adaptors by exponential amplification of these products. The optimized cycles for the first and second PCR were 27 and 13 respectively to increase representation and reduce redundancy of subtracted cDNA libraries. Secondary PCR products were used as templates for PCR amplification of human G3PDH at 20, 25, 30 and 35cycles to assure subtraction efficiency. PCR products were run on 1.8% agarose gel. Products of the secondary PCR reactions were Cloned into a pT Adv vector (CLONTECH) and the resultant ligation products were then transformed into DH5 a E. co...