To Construct A CDNA Library From Human Hepatocellular Carcinoma Tissue

Objective:To construct a cDNA library from human hepatocellular carcinoma tissue.Methods:The total RNA was extracted from human HCC (hepatocellular carcinoma tissue)and the mRNA was isolated from the total RNA by MagneSphere technique,and then the first-stand cDNA was synthesized with oligo(dT)primer containing sfiⅠsite while the double-strand cDNA was amplified through LD-PCR(long-distance PCR) by SMART technique.The double strand cDNA was digested by sfiⅠ(ⅠA&ⅠB)restriction enzyme before cDNA size fractionation,and the double-strand cDNA fractionated was ligated into theλTripIEX2 vector and then was packaged intoλphage in vitro.Titering the unamplified library,and determining the percentage of recombinant clones.Then, screening cDNA insert by PCR to test for ligation efficiency.It was amplified and identified.At last,the library was screened with the serum of hepatocellular carcinoma patient and sequence of the reacted clones were determined.Results:The unamplified library consists of 6.9×10~6 pfu/ml,and the percentage of recombinants clones was 97.6%.The titer of the amplified cDNA library was 1.7×10~9 pfu/ml and the average insert of the recombinants was 1.3kb.7 positive clones were obtained after three cycles screening,and the sequences were determined and analyzed with BLAST software in GenBank.As a result,positive clones were derived frome different genes.All Of genes were high homologous to genes known in GenBank,and there were also genes with low homology to genes kown in GenBank.Conclusions:The quality of the constructed cDNA library from human HCC tissue is excellent and helpful for futher research.Positive clones were obtained after screening,and of it maybe novel genes.The associations of these genes to colorectal carcinoma and the roles that they play should be revealed in the future.