Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma.
OBJECTIVE
To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database.


METHODS
Human VECs related to gastric adenocarcinoma and corresponding normal tissue were separated by magnetic beads coupled with antibody CD31 (Dynabeads CD31). A few amount of total RNA were synthesized and amplified by SMART PCR cDNA Synthesis Kit. Then, using SSH and T/A cloning techniques, cDNA fragments of differentially expressed genes in human VECs of gastric adenocarcinoma were inserted into JM109 bacteria. One hundred positive bacteria clones were randomly picked and identified by colony PCR method. To analyze cDNA libraries of gastrocarcinoma and VECs in CGAP database, the tools of Library Finder, cDNA xProfiler, Digital GENE Expression Displayer (DGED), and Digital Differential Display (DDD) were used.


RESULTS
Forward and reverse subtraction cDNA libraries of human VECs related to gastrocarcinoma were constructed successfully with SSH and T/A cloning techniques. Analysis of CGAP database indicated that no appropriate library of VECs related to carcinoma was constructed.


CONCLUSIONS
Construction of subtraction cDNA libraries of human VECs related to gastrocarcinoma was successful and necessary, which laid a foundation for screening and cloning new and specific genes of VECs related to gastrocarcinoma.
Liu
Yong-Bo
YB
Department of Cell Biology and Medical Genetics, Medical College of Zhengzhou University, Zhengzhou, 450052, Henan Province, China. liuyb415@163.net
Wei
Zhao-Xia
ZX
Li
Li
L
Li
Hang-Sheng
HS
Chen
Hui
H
Li
Xiao-Wen
XW
D018076:DNA, Complementary / Q000032:analysis; D015252:Deoxyribonucleases, Type II Site-Specific; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic; D015723:Gene Library; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics*
D018076:DNA, Complementary; D015252:Deoxyribonucleases, Type II Site-Specific; C040158:GTAC-specific type II deoxyribonucleases