| Gene chip or DNA microarray is a newly developed technology combining molecular biology, microelectronics, physics, chemistry, and other sophisticated approaches. DNA microarray technology began in early the 1990s, has been developing quickly in recent years and has been applied to genomics, post-genomics and other biotechnology studies. However DNA microarray has many aspects to be optimized, so as to be applied more widely in both research areas and in clinical applications. This study addresses the optimization some conditions of microarray technique, while studying a cellular model of gene expression profile.The mechanism of neuron differentiation is still unknown. All-trans Retinoic Acid (RA) is one of the tumor chemotherapy agents, which can induce SH-SY5Y cell to differentiate into neuron-like state and inhibit the SH-SY5Y cellular growth. This process is a classic model in the research of neuron differentiation. Recent studies indicate that the mechanism to this process is complicated and needs further exploration with sophisticated microarray approaches. On the bases of this model, we prepared cDNA microarray with Restriction Display (RD) technique and studied the gene expression profile changes of SH-SY5Y cells after treatment with RA with our self-made microarrays.The researches address the following aspects:1. cDNA fragments library construction of SH-SY5Y cells using RD technique. Big amount of gene probes collection has been regarded as the prerequisite for the preparation of DNA microarrays. In the process of library construction, some technical details were studied, such as the combination of DNA silver staining with RD technique, purification of PCR product, storage of the data about probes and bioinformatics analysis of one EST, etc.2. Study on the preparation of DNA microarray: In this study, conditions influencing the hybridization of gene chip, re-use of a gene chip andpost-printing examination of the gene chip were studied in details.3. Application of microarray on the differentiation of SH-SY5Y cells induced by RA. SH-SY5Y cells were differentiated into neuron-like cells by RA treatment in low concentration, total RNA was extracted and labeled to hybridize with the microarray, the changes on gene expression were detected to find differentially expressed genes. Results:1 .Establishing a method to separate gene fragments, combining with the RD technique with DNA silver staining, the results showed that in the PAGE gel, every group contained 7-10 clear DNA bands on average. The DNA bands separated from the gel might be specifically amplified, and the products could be ligated into the vectors. These results indicate that silver staining-RD technique could be efficiently used to quickly isolate EST fragments in visible light.2. PCR products can be purified by CTAB method, which removing the dNTP and primers. The purified products can be used in the ligation reaction to pMD18-T vector with high efficiency.3. To establish gene fragments library by RD technique has the advantages of simple, efficient and low redundancy. By this way, there were altogether 1078 gene fragments isolated in our experiment and the DNA sequencing showed high fidelity.4. The data of probes were easily collected, saved and regulated by FOXPRO software, with further improvements.5. One gene fragment was separated by RD technique and analyzed with bioinformatics indicating that the EST comes form a novel gene.6. The gene fragments prepared by RD technique were successfully used in study the gene expression profile of the SH-SY5Y. The gene fragments length ranged from 250bp to 750bp with nearly the same hybridization temperature and so the hybridization conditions were easy standardized.7. The labeled sample was purified and hybridized with the self-made cDNA microarray, which demonstrated low background and clear positive signals.8. The gene chip made with amino-modified glass can be reused, after wash by stripping solution to remove the probes, satisfactory results can...
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