The Unigene incorporates hundreds of thousands of sequences of expressed sequence tag (EST) whose tissue distributions are labelled (Schuler, 1997). cat ttc ctc agc att tac. The amplified products were digested with BL-21 and the recombinant ZNF165 protein accounted for 15% of total protein. After the induction with 1?mM IPTG at 37C for 6?h, the recombinant ZNF165 protein fused with 6 His tag was purified by Ni2+ affinity chromatography. Western blotting Purified recombinant ZNF165 protein was separated on 10% SDSCpolyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. After blocking with the TNT solution containing 5% dry milk, the membranes were incubated with sera from normal individuals or HCC patients at a 1?:?500 dilution for 1.5?h, and then with goat anti-human IgG (H+L) alkaline phosphatase conjugate (Promega) for 1?h. Colour substrate NBT/BCIP (Promega) was added and incubated for 10?min for colour development. RESULTS Databases analysis Unigene Leucovorin Calcium is an experimental processing system for automatically partitioning GenBank sequences into a nonredundant set of gene-oriented clusters. Each Unigene cluster contains sequences that represent a Unigene, as well as the related information such as the tissue types in which the gene is expressed. The Unigene incorporates Leucovorin Calcium hundreds of thousands of sequences of expressed sequence tag (EST) whose tissue distributions are labelled (Schuler, 1997). SAGE is an on-line technique that allows quantitation and comprehension of cellular gene expression profiling (Velculescu em et al /em , 1995). Consequently, Unigene and SAGE can be used to analyse the spatial and temporal expression pattern of a given gene and predict the functional information of the gene. In the database of Unigene, there were 25 ESTs representing ZNF165. Although 14 of these ESTs were identified in the libraries of normal tissues such as the testis and placenta (10), pancreatic islet (3) and breast (1), the remainder 11 ESTs were derived from several types of tumours, such as uterus tumour, well-differentiated endometrial adenocarcinoma, leiomyosarcoma, germ cell tumour and carcinoid. In the SAGE database, one tag (AGGGAAAACC) representing ZNF165 was identified from the libraries of mammary gland carcinoma, prostate carcinoma and pancreas adenocarcinoma, respectively. These data suggest that ZNF165 mRNA has the expression characteristics typical of a CT antigen gene. Expression profile of ZNF165 mRNA One of the criteria for identifying CT antigen genes is their specific expression in tumours, but not in normal tissues except the testis. The mRNA expression profile of ZNF165 was determined by RTCPCR. Leucovorin Calcium A series of tissues were examined, including normal tissues, HCC, gastric, colon and non-small-cell lung cancers and paired noncancerous tissues. mRNA expression of ZNF165 was first examined by RTCPCR with the amplification by 30 cycles in 16 different normal tissues including the spleen, prostate, testis, ovary, small intestine, colon, peripheral blood, heart, lung, liver, whole brain, kidney, pancreas, placenta, thymus and skeletal muscle. ZNF165 mRNA expression was detected only in the testis (Figure 1A). However, quantification of gene expression by real-time PCR with the amplification of 40 cycles revealed that ZNF165 mRNA could also be detected in the placenta, lung, liver, pancreas, spleen, thymus and colon, but not in other tissues. The average EI values of ZNF165 mRNA expression in the testis and other normal tissues including the placenta, lung, liver, pancreas, spleen, thymus and colon are 33.56 and 0.24, respectively. Therefore, the expression level of ZNF165 mRNA in these normal tissues was 140-fold lower than that in the testis (Figure 2). Open in a separate window Figure 1 Expression patterns of ZNF165 mRNA in 16 normal tissues, HCC Rabbit polyclonal to ZFHX3 and paired noncancerous liver tissues. (A) Gel electrophoresis of RTCPCR products amplified by 30 cycles shows ZNF165 mRNA expression in 16 normal tissues. ZNF165 mRNA is only expressed in testis. 1C16 normal tissues: 1. brain; 2. heart; 3. kidney; 4. liver; 5. lung; 6. pancreas; 7. placenta; 8. skeletal muscle; 9. prostate; 10. ovary; 11. leucocyte; 12. testis; 13. small intestine; 14. spleen; 15. colon; 16. thymus. (B) Gel electrophoresis of RTCPCR products amplified by 30 cycles shows ZNF165 mRNA in the paired HCC and noncancerous liver tissue samples. Leucovorin Calcium RTCPCR for G3PDH was used to monitor the quality of the mRNA samples. Ca: HCC; N: paired noncancerous liver tissues. Open in a separate window Figure 2 Quantification of ZNF165 mRNA expression in 22 HCC tissues, eight noncancerous liver tissues and normal tissues including the testis, placenta, lung, liver, pancreas, spleen, thymus and colon. Hepatocellular carcinoma: the average EI value is 22.81 in 22.