|
| Status |
Public on Sep 29, 2007 |
| Title |
MDA-MB-231_TspR1-ExoIII-rep1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
MDA-MB-231-MOCK
|
| Organism |
Homo sapiens |
| Characteristics |
Cell line: MDA-MB-231
Gender: Female
Age: 51
Tissue: Breast tumor
|
| Treatment protocol |
Genomic DNA was first digested with TspR I, which created DNA fragments with 9-base 3’ extension and were resistant for exonuclease III digestion. The DNA were NOT digested with methylation-sensitive restriction enzyme Hpa II. Then treatment with exonuclease III and RecJf exonuclease, DNA were purified and labeled with Cy3 and Cy5 fluorescence dyes respectively. The Cy3 hybridization intensity was normalized to Cy5 for comparison among samples.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were grown to 90 % confluence in 10 cm tissue culture dish. Each 10 cm dish was washed with 1X PBS for three times, and resuspended with 700μl of cell lysis buffer. Suspensions were collected in a 1.5ml eppendorf tube and treated with 0.1 mg/ml of RNase A for an hour at 37 ℃ and then with 0.3 mg/ml of proteinase K for 12-16 hours at 50℃. An equal volume of phenol/chloroform/isoamyl alcohol mixture (24:25:1) was added, mixed thoroughly, and centrifuged at 13000 rpm for 10 min. The aqueous phase was collected carefully, and then repeated the extraction procedure until the interface is clean. The aqueous phase again was collected and an equal volume of chloroform was added, mixed thoroughly, and centrifuged at 13000 rpm for 10 min. This procedure was repeated until the intersurface was clean. Finally, total genomic DNA in the aqueous phase was precipitated by the addition of 1/10 volume of 7.5 M ammonium acetate (pH: 5.2) and two volume of absolute ethanol. The mixture was incubated at -70℃ overnight and centrifuged at 13000 rpm, 4℃ for 30 min. Supernatant was removed and the pellet was washed by proper amount of 70 % ethanol and then centrifuged. In the end, the pellet was redissolved in d3H20 and then stored at -20℃. The integrity of the DNA extracted was checked by 1.2 % (w/v) agarose gel electrophoresis. The concentration of DNA was estimated by ultraviolet spectrophotometry.
|
| Label |
Cy3
|
| Label protocol |
According to the manual of Agilent Oligo Microarray Kit.
|
| |
|
| Channel 2 |
| Source name |
MDA-MB-231-HpaII
|
| Organism |
Homo sapiens |
| Characteristics |
Cell line: MDA-MB-231
Gender: Female
Age: 51
Tissue: Breast tumor
|
| Treatment protocol |
Genomic DNA was first digested with TspR I, which created DNA fragments with 9-base 3’ extension and were resistant for exonuclease III digestion. The DNA were then digested with methylation-sensitive restriction enzyme Hpa II. After treatment with exonuclease III and RecJf exonuclease, DNA were purified and labeled with Cy3 and Cy5 fluorescence dyes respectively. The Cy3 hybridization intensity was normalized to Cy5 for comparison among samples.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were grown to 90 % confluence in 10 cm tissue culture dish. Each 10 cm dish was washed with 1X PBS for three times, and resuspended with 700μl of cell lysis buffer. Suspensions were collected in a 1.5ml eppendorf tube and treated with 0.1 mg/ml of RNase A for an hour at 37 ℃ and then with 0.3 mg/ml of proteinase K for 12-16 hours at 50℃. An equal volume of phenol/chloroform/isoamyl alcohol mixture (24:25:1) was added, mixed thoroughly, and centrifuged at 13000 rpm for 10 min. The aqueous phase was collected carefully, and then repeated the extraction procedure until the interface is clean. The aqueous phase again was collected and an equal volume of chloroform was added, mixed thoroughly, and centrifuged at 13000 rpm for 10 min. This procedure was repeated until the intersurface was clean. Finally, total genomic DNA in the aqueous phase was precipitated by the addition of 1/10 volume of 7.5 M ammonium acetate (pH: 5.2) and two volume of absolute ethanol. The mixture was incubated at -70℃ overnight and centrifuged at 13000 rpm, 4℃ for 30 min. Supernatant was removed and the pellet was washed by proper amount of 70 % ethanol and then centrifuged. In the end, the pellet was redissolved in d3H20 and then stored at -20℃. The integrity of the DNA extracted was checked by 1.2 % (w/v) agarose gel electrophoresis. The concentration of DNA was estimated by ultraviolet spectrophotometry.
|
| Label |
Cy5
|
| Label protocol |
According to the manual of Agilent Oligo Microarray Kit.
|
| |
|
| |
| Hybridization protocol |
According to the manual of Agilent protocol (http://www.agilent.com)
|
| Scan protocol |
Scanning was performed as the manual of Agilent protocol (http://www.agilent.com)
|
| Description |
Protected from Exonuclease III digestion by TspRI ends.
1~2μg DNA was digested with methylation sensitive restriction enzyme in 10μl of total volume for 2hrs, 1μl(10units) TspR I was then added and reacted at 65℃ for two hours using hot top PCR machine. 10unit of Exonuclease III enzyme was then added and total volume was brought to 20μl by addition of water and 10X buffer. Reaction was carried out at 30℃ for 1hr. Exonuclease III was heat inactive by 70℃ for 20min. 30units RecJf was added to remove single-strand DNA and the enzyme was inactivated by heating at 65℃ for 20min. DNA was then phenol/chloroform extracted and ethanol precipitate.
|
| Data processing |
The Cy3 hybridization intensity was normalized to Cy5 for comparison among samples. The log2 ratio(log2 Cy5/Cy3) were calculated and compared. Agilent software was used.
|
| |
|
| Submission date |
Aug 31, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Ming-Ta Hsu |
| E-mail(s) |
d88307@ym.edu.tw
|
| Phone |
886-2-28267230
|
| Organization name |
National Yang-Ming University
|
| Street address |
155, Li-Nong St., Sec.2, Peitou, Taipei, Taiwan, R.O.C.
|
| City |
Taipei |
| ZIP/Postal code |
112 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL4091 |
| Series (1) |
| GSE9015 |
Genome-Wide Mapping of Hypomethylated Sites in Human Genomes |
|
