Title page
Acknowledgement
Contents
Abstract 12
I. Introduction 14
II. Materials and Methods 17
II.1. Small interfering BRCA1 (siRNA) expression vector 17
II.2. Human samples, cell lines, culture conditions, and transfection 17
II.3. Total RNA isolation and quantitative real-time RT-PCR 18
II.4. Western blot 18
II.5. DNA isolation and bisulfite modification 19
II.6. cDNA Microarray 19
II.7. Pyrosequencing 20
III. Results and Discussion 21
III.1. BRCA1 knockdown by small interfering RNA 21
III.2. The expression pattern of XIST by BRCA1-siRNA in MCF7 21
III.3. The expression pattern of X-linked genes by BRCA1-siRNA 22
III.4. Quantification of methylation percentage of the 5' -end of XIST in serum by pyrosequencing 24
III.5. Decreased methylation percentage of the 5 -end of XIST by treatment with a DNA methylation inhibitor 25
III.6. Determination of the accuracy of pyrosequencing 26
III.7. XIST RNA expression correlates with its methylation status 26
Reference 40
Abstract in Korean 44
Table 1. Quantitative real-time RT-PCR conditions for gene expression analysis 28
Table 2. List of the X-linked oncogenes that were differently expressed by 2-fold or greater 29
Figure 1. Suppression of BRCA1 protein by pSUPER.retro.puro-BRCA1 in MCF7. 30
Figure 2. Expression pattern of XIST by BRCA1-siRNA. 31
Figure 3. Expression pattern of X-linked genes by BRCA1-siRNA. 32
Figure 4. The design for methylation analysis of the 5' -end of XIST and its quantitation by pyrosequencing. 34
Figure 5. Decrease of the methylation percentage of the 5' -end of XIST by the treatment of DNA methylation inhibitor and determination of its accuracy. 35
Figure 6. Pyrosequencing data of the 5' -end of XIST methylation and its gene expression in human cell lines and tissues. 36