中文摘要
本論文主要架構可分成三大部分，第一部份為本實驗室cDNA 微陣列系統的介紹，詳細介紹硬體及軟體的詳細測試數據及應用原理，並介紹實驗流程的詳細步驟。第二部份為介紹本實驗室基因微陣列系統之優化相關研究，包括探針DNA純化方式測試、Total RNA反轉錄標定螢光染劑之測試、利用GAPDH基因觀察螢光標的物雜合情形之測試、雜合反應後分析影像出現循環週期訊號之探討。此部份研究的重點在尋找出本實驗室基因微陣列系統的重要相關參數值，以利於後續研究者之參考。第三部份為利用基因微陣列系統觀察九種細胞凋亡基因表現情形。此部份是將Jurkat細胞照射20Gy γ射線後，於照射後2、4、6、8、12小時五個時間點中，分別取出實驗組及對照組Jurkat細胞的Total RNA，經由反轉錄方式分別標定Cy3和Cy5兩種螢光染劑，再與載玻片中九種連續排印300點的細胞凋亡基因做雜合反應，經由共軛焦雷射掃描系統讀出相對應的螢光強度值，再利用數學統計方式觀察九種基因在五個時間點中基因表現情形。

關鍵字：cDNA微陣列，反轉錄，Jurkat細胞，基因表現，細胞凋亡。

Abstract
This thesis contains three main parts. In the first part we provide the introduction to cDNA microarray system. We also detail the testing data and applied theory of either hardware or software and each step of experimental procedures. In the second part we will introduce our related research about the optimization of cDNA microarray system in our lab including tests of probe DNA purification, tests of fluorescence labeled total RNA reverse transcription, tests of observing conditions of hybridization fluorescent targets by utilizing GAPDH gene. And we explore the cycling signals of analyzed images after hybridization. The key point in this part of research is to find out the important related parameters of our cDNA microarray system as the references for following studies. The third part is on utilizing cDNA microarray system to observe gene performance of Apoptosis. We let the Jurkat cells be irradiated by gamma rays for 20Gy and extract the total RNA of Jurkat cells at five time points 2, 4, 6, 8, 12 hours respectively after being irradiated and they are labeled two kinds of dyes, Cy3 and Cy5 individually via reverse transcription. These labeled RNA hybridize with 300 points of apoptosis genes arranged in order on the slide. We observe the gene expression of these nine kinds of genes at five different time points via using statistical method to analyze the corresponding fluorescent intensity read out from confocal laser scanning system.

Keyword: cDNA microarray, reverse transcription, Jurkat cell, gene expression, apoptosis.

參考文獻
1. Brown PO, Botstein D, Exploring the new world of the genome with DNA microarray. Suppl Nat Genet. 21:33-37,1999.
2. Christine E. Canman, Michael B. Kastan, Induction of apoptosis by tumor suppressor genes and oncogenes. Cancer Biology 6:17-25,1995.
3. Debouck C. Goodfellow PN, DNA microarrays in drug discovery and development. Nature genet. 21:48-50,1999.
4. Duggan DJ, Bittner M, Chen Y, Meltzer P, Trent JM, Expression profiling using cDNA microarrays. Nature genet. 21:10-14, 1999.
5. Eisen MB, Spellman PT, Brown PO, Bostein D, Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA. 95:14863-14868,1998.
6. Evans T, Rosenthal ET, Youngblom J, Distel D, Hunt T, Cyclin: A protein specified by material mRNA in sea urchin eggs that is destroyed at each cleavage division. Cell 33:389,1983.
7. Halgren RG, Fielden MR, Fong CJ, Zacharewski TR, Assessment of clone identity and sequence fidelity for 1189 IMAGE cDNA clones. Nucleic Acids Research 29:582-588,2001.
8. Hallahan DE, Radiation-mediated gene expression in the pathogenesis of the clinical radiation response. Semin. Radiat. Oncol. 6:250-267,1996.
9. Hartwell LH, Weinert TA, Checkpoints: Controls that ensure the order of cell cycle events. Scinece 246:629,1989.
10. Heller RA, Davis RW, Discovery and analysis of inflammatory disease-related genes using cDNA microarray. Proc. Natl. Acad. Sci. USA. 94:2150-2155,1997.
11. Hengartner MO, Ellis RE, Horvitz HR, Caenorhabditis elegans gene ced-9 protects cells form programmed cell death. Nature 356:494,1992.
12. Hong JH, Gatti RA, Hou YK, Chiang CS, McBride WH, G2/M-phase arrest and release in ataxia telangiectasia and normal cells after exposure to ionizing radiation. Radiation Research 140:17-23,1994.
13. http://cmgm.stanford.edu/pbrown/protocols/ASH/pcrcleanupcomparison.htm
14. http://cmgm.stanford.edu/pbrown/mguide/tips.html
15. Jarvis WD, Kolesnick RN, Fornari FA, Traylor RS, Gewirtz DA, Grant S, Introduction of apoptosis DNA damage and cell death by activation of the sphingomyelin pathway. Proc. Natl. Acid Sci. USA. 74:777,1994.
16. Kerr JF, A histochemical study of hypertrophy and ischaemic injury of rat liver with special reference to changes in lysosomes. J Pathol Bacteriol 90:419,1965.
17. Khan J, Bittner ML, Chen Y, Meltzer PS, Trent JM, DNA microarray technology: the anticipated impact on the study of human disease. Biochimica et Biophysica Acta 1423:M17-M28, 1999.
18. Kirschner M, The cell cycle then and now. Trends Biochem Sci. 17:281,1992.
19. Korsmeyer SJ, Bcl-2: A repressor of lymphocyte death. Immunol Today 13:285,1992.
20. Kristina AC, David BK, Michael R, The genetics of cancer—a 3D model. Nature genet. 21:38-41,1999.
21. Lipshutz RJ, Fodor SPA, Gingeras TR, Lockhart DJ, High density synthetic oligonucleotide arrays. Nature genet. 21:20-24,1999.
22. Martin SJ, Green DR, Cotter TG, Dicing with death: Dissecting the components of the apoptosis machinery. Trends Biochem Sci. 19:26,1994.
23. Mitchison JM, The biology of cell cycle. Camgridge University Press, New York, 1971.
24. Murray AW, Kirschner MW, Dominoes and clocks: The union of two views of the cell cycle. Science 246:614,1989.
25. Muzio M., Signalling by proteolysis: death receptors induce apoptosis. Int. J. Clin. Lab. Res. 28: 141–147,1998.
26. Nurse P, Bisset Y, Gene required in G1 for commitment to cell cycle and in G2 for control of mitosis in fission yeast. Nature 292:558,1981.
27. Pastinen T, Raitio M, Lindroos K, Tainola P, Peltonen L, Syvanen AC, A system for specific, high-throughput genotyping by allel-specific primer expression on microarray. Genome Research 10:1031-1042,2000.
28. Perou CM, Jeffrey SS, Rijn M, Shalon D, Brown PO, Botstein D, Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc. Natl. Acad. Sci. USA. 96:9212-9217,1999.
29. Schena M, Shalon D, Heller R, Chai A, Brown PO, Davis RW, Parallel human genome analysis: Microarray-based expression monitoring of 1000 genes. Proc. Natl. Acad. Sci. USA. 93:10614-10619,1996.
30. Sherlock G, Hernandez-Boussard T, Kasarskis A, Binkley G, Matese JC, Dwight SS, Kaloper M, Weng S, Jin H, Ball CA, Eisen MB, Spellman PT, Brown PO, Botstein D, Cherry JM, The stanford microarray database. Nucleic. Acids Research, 29:152-155,2001.
31. Sally AA, Khanh TD, Sohrab S, Michael B, Paul M, Jeffrey T, Albert JF, Identification of potential mRNA Biomarkers in peripheral blood lymphocytes for human exposure to ionizing radiation. Radiation Research 154:324-346, 2000.
32. Tanaka TS, Minoru SHK, Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray. PNAS. 97:9127-9132,2000.
33. Wang K, Gan L, Jeffery E, Gayle M, Gown AM, Skelly M, Nelson PS, NG WV, Schummer M, Hood L, Mulligan J, Monitoring gene expression profile changes in ovarian carcinomas using cDNA microarray. Gene 299:101-108,1999.