|
| Status |
Public on Aug 31, 2009 |
| Title |
CpG island methylation in KATOIII |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Sonicated KATOIII genomic DNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: gastric cancer cell line, KATOIII
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA lysis buffer plus proteinase K. phenol/chloroform extraction. Ethernol precipitated. RNase treated.
|
| Label |
Cy3
|
| Label protocol |
One µg of DNA was labelled using Agilent Genomic DNA Labeling Kit PLUS (5188-5309).
|
| |
|
| Channel 2 |
| Source name |
MeDIP KATOIII
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: gastric cancer cell line, KATOIII
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA lysis buffer plus proteinase K. phenol/chloroform extraction. Ethernol precipitated. RNase treated.
|
| Label |
Cy5
|
| Label protocol |
Five µg of genomic DNA fragmented by sonication was immunoprecipitated with five µg of anti-5-methyl cytidine antibody (Diagnode) and solved in 50 µL of buffer. Immunoprecipitated DNA was checked by real-time PCR to ensure that it was enriched. Fourty-five µL of DNA immnoprecipitated was labelled using Agilent Genomic DNA Labeling Kit PLUS (5188-5309).
|
| |
|
| |
| Hybridization protocol |
Labelled DNA mixed with Cot-1 DNA and Agilent blocking solution was hybridized to arrays for 40 hours at 67C and 10rpm. Arrays were washed as per Agilent protocol.
|
| Scan protocol |
Arrays were washed and scanned immediately with Agilent microarray scanner.
|
| Description |
To recover methylated DNA with anti-5-methyl cytidine antibody, DynaBeads protein A (Dynal) was used.
|
| Data processing |
Arrays were processed using Agilent Feature Extraction software and normalized (Background subtraction) using Agilent Chip Analytics software. Our original parameter, "Me value" as a value that linearly correlates with the fraction of methylated DNA molecules in CpG islands in samples with various overall DNA methylation levels, was calculated from subtracted signal log2 ratio and p[Xbar].
Methylation statuses were defined according to Me_Value. U, <0.4; M, >0.6; -, between 0.4 and 0.6. x, Probes that showed Cy3 signal intensities (Input) more than five times average signal intensity. The probes were eliminated as non-functional probes due to high possibility of cross-hybridization.
|
| |
|
| Submission date |
Mar 19, 2009 |
| Last update date |
Mar 19, 2009 |
| Contact name |
Satoshi Yamashita |
| E-mail(s) |
syamashi@maebashi-it.ac.jp
|
| Organization name |
National Cancer Center Research Institute
|
| Street address |
Tsukiji 5-1-1
|
| City |
Chuo-ku |
| State/province |
Tokyo |
| ZIP/Postal code |
104-0045 |
| Country |
Japan |
| |
|
| Platform ID |
GPL4126 |
| Series (1) |
| GSE15291 |
Development of a novel parameter for quantification of methylation levels in MeDIP-CpG island microarray analysis |
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