Objective: To obtain differentially expressed genes related to human glioma using cDNA microarray and to characterization of one novel full-length gene.
Method: Four samples of human glioma samples, 1 fetal brain tissue sample, and 2 normal brain tissue samples were used to extract the total RNA, and the mRNA was used to make probes. After hybridization and washing procedure, the products of hybridization were scanned using computer system. One gene, named 436F11 clone, was subsequently analyzed by Northern blotting, bioinformatics, and protein expression.
Result: Fifteen differentially expressed new genes related to human glioma were obtained through four times of hybridization and scanning. Northern blotting confirmed that over-expression of 436F11 gene in the normal human brain tissue and low-expression in the human glioma tissues. The analysis of BLASTn and BLASTx showed that the clone of 436F11 was a novel full-length gene coding 78 amino acids of protein with a theoretical relative molecular weight of 8648 and an isoelectric point of 4.69 and that it was 60% identical to mouse PKIbeta amino acid, so it was called human PKIbeta gene. After it was transfected into Escherichia coli, higher-expressed protein of PKIbeta was obtained which yielded a major clear band on an SDS-PAGE gel after purification. The products obtained from amino acid sequencing and molecular weight detection were exactly the same as the products performed by bioinformatic analysis.
Conclusion: cDNA microarray technology can be successfully applied to identify differentially expressed genes. PKIbeta may be a novel full-length gene related to human glioma and may provide a new way to gene therapy of glioma.