|
| Status |
Public on Dec 28, 2012 |
| Title |
Gastric cancer 12T |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
MMASS_methylated DNA
|
| Organism |
Homo sapiens |
| Characteristics |
Sex: female
age: 63
survival: Long (> 5 years)
|
| Treatment protocol |
Not applicable
|
| Growth protocol |
Not applicable
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was digested using MseI then ligated to linker. For representation of methylated sequences, the sample was restricted by a combination of BstUI, HhaI and HpaII. Then a linker PCR was carried out using the adaptor ligated to the MseI fragments. For representation of unmethylated sequences, subtraction hybrididzation was used to remove an amplicon of methylated sequences.
|
| Label |
Cy5
|
| Label protocol |
300 ng of PCR product together with 0.5 ng of control Arabidopsis thaliana cDNA (synthesized from pARAB obtained from the Microarray Centre, University Health Network, Toronto, Canada) were indirectly labeled using aminoallyl-dUTP and the the BioPrime Labeling Kit (Invitrogen). 300 ng of PCR product together with 0.5 ng of control Arabidopsis thaliana cDNA (synthesized from pARAB obtained from the Microarray Centre, University Health Network, Toronto, Canada) were indirectly labeled using aminoallyl-dUTP and the the BioPrime Labeling Kit (Invitrogen).
|
| |
|
| Channel 2 |
| Source name |
MMASS_unmethylated DNA
|
| Organism |
Homo sapiens |
| Characteristics |
Sex: female
age: 63
survival: Long (> 5 years)
|
| Treatment protocol |
Not applicable
|
| Growth protocol |
Not applicable
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was digested using MseI then ligated to linker. For representation of methylated sequences, the sample was restricted by a combination of BstUI, HhaI and HpaII. Then a linker PCR was carried out using the adaptor ligated to the MseI fragments. For representation of unmethylated sequences, subtraction hybrididzation was used to remove an amplicon of methylated sequences.
|
| Label |
Cy3
|
| Label protocol |
300 ng of PCR product together with 0.5 ng of control Arabidopsis thaliana cDNA (synthesized from pARAB obtained from the Microarray Centre, University Health Network, Toronto, Canada) were indirectly labeled using aminoallyl-dUTP and the the BioPrime Labeling Kit (Invitrogen). 300 ng of PCR product together with 0.5 ng of control Arabidopsis thaliana cDNA (synthesized from pARAB obtained from the Microarray Centre, University Health Network, Toronto, Canada) were indirectly labeled using aminoallyl-dUTP and the the BioPrime Labeling Kit (Invitrogen).
|
| |
|
| |
| Hybridization protocol |
Labelled DNA was purified using a Qiaquick PCR purification column and then vacuum dried. Both dye-coupled DNAs were then resuspended together in 85 ul of DIG Easy Hyb solution (Roche Diagnostics) together with 5 ul of salmon sperm DNA (10 ug/ul) and denatured at 95 C for 5 min. The hybridization mixture was allowed to cool briefly and 5 ul of yeast tRNA (10 ug/ul) was added and the mixture was held at 65 C for 2 min and allowed to cool to room temperature. Hybridization to the microarray was carried out under a cover-slip in a humidified chamber at 37 C for 8 h. The cover slip was floated off in 1 x SSC and each slide was washed three times in 1x SSC, 0.1% SDS at 50 C for 15 min followed by removal of SDS at room temperature in 1 x SSC and 0.1 x SSC for 5 min each.
|
| Scan protocol |
The slides were dried by centrifugation at 500 r/min for 5 min and scanned immediately using the GenePix 4000A scanner (Axon). The settings for PMT gain were adjusted during the initial rapid scan to achieve a balance between the two channels and these settings were used for the high resolution scan. GenePix version 4.1 was used to perform image analysis and feature segmentation.
|
| Data processing |
Normalisation was performed using a subset of high-intensity methylated clones that demonstrated consistent log-ratios between replicate arrays.
|
| |
|
| Submission date |
Jul 24, 2012 |
| Last update date |
Dec 28, 2012 |
| Contact name |
Alfred S Cheng |
| E-mail(s) |
[email protected]
|
| Organization name |
Institute of Digestive Disease, The Chinese University of Hong Kong
|
| Street address |
Rm 707, LiKaShing Medical Sciences Building, Prince of Wales Hospital, Shatin
|
| City |
Hong Kong |
| ZIP/Postal code |
852 |
| Country |
Hong Kong |
| |
|
| Platform ID |
GPL2040 |
| Series (2) |
| GSE39599 |
Helicobacter pylori-mediated Epigenetic Dysregulation of FOXD3 Tumor-suppressive Cascade In Gastric Carcinogenesis [MMASS] |
| GSE39600 |
Helicobacter pylori-mediated Epigenetic Dysregulation of FOXD3 Tumor-suppressive Cascade In Gastric Carcinogenesis |
|
