|
| Status |
Public on Jan 22, 2010 |
| Title |
MeDIP 1st antibody incubation time 12h, 2nd antibody incubation time 1h - 803 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Input (methylated + unmethylated) DNA of gastric adenocarcinoma cell line MGC-803
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: mucinous adenocarcinoma, poorly differentiated, highly tumorigenesis, endepidermis, adherence, Han people, male; 1st antibody incubation time 12h, 2nd antibody incubation time 1h; methylated + unmethylated DNA
cell line: MGC-803
|
| Growth protocol |
Gastric epithelium cell line Ges-1 and the gastric adenocarcinoma cell line MGC-803 was grown in 10×10cm dish with RPMI1640 medium and the temperature maintained at 37°C. Medium was used with FPS (10%) and penicillin-Streptomycin (1%).CO2 levels were monitored at 5.0% ±0.2%.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After harvested, the genomic DNA were extracted by NucleoSpin Tissue kit(MN ).
|
| Label |
Cy3,Cy5
|
| Label protocol |
Labelled with Cy3 and Cy5 during PCR the methylated and unmethylated fragment as manufacturers instructions. Samples purified using QIAGEN PCR purification kit before hybridization.
|
| |
|
| Channel 2 |
| Source name |
IP (methylated) DNA of gastric adenocarcinoma cell line MGC-803
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: mucinous adenocarcinoma, poorly differentiated, highly tumorigenesis, endepidermis, adherence, Han people, male; 1st antibody incubation time 12h, 2nd antibody incubation time 1h; methylated DNA
cell line: MGC-803
|
| Growth protocol |
Gastric epithelium cell line Ges-1 and the gastric adenocarcinoma cell line MGC-803 was grown in 10×10cm dish with RPMI1640 medium and the temperature maintained at 37°C. Medium was used with FPS (10%) and penicillin-Streptomycin (1%).CO2 levels were monitored at 5.0% ±0.2%.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After harvested, the genomic DNA were extracted by NucleoSpin Tissue kit(MN ).
|
| Label |
Cy5,Cy3
|
| Label protocol |
Labelled with Cy3 and Cy5 during PCR the methylated and unmethylated fragment as manufacturers instructions. Samples purified using QIAGEN PCR purification kit before hybridization.
|
| |
|
| |
| Hybridization protocol |
Hybridization of the CGI array was performed under a cover slip in a humidified chamber fixed in a BioMix II hybridization machine (CapitalBio, China) at 42°C for 17 h. The array was washed two times in 2X saline sodium citrate and 0.2% SDS at 42°C for 5 min and once in 0.1X saline sodium citrate at room temperature. The slides were dried by centrifugation at 800 rpm for 5 min and scanned immediately with LuxScan 10K scanner (CapitalBio, China).
|
| Scan protocol |
Arrays scanned using an LuxScan 10K scanner and LuxScan 10K software (CapitalBio, China).
|
| Description |
MeDIP with 1st antibody incubation time 12h, 2nd antibody incubation time 1h using gastric adenocarcinoma cell line MGC-803 to evaluate antibody incubation time
|
| Data processing |
The median average intensity of foreground and background was extracted from the .lsr files. If a spot intensity was zero or negative after background subtraction, it was set at half of the minimum positive corrected intensities in the array. We performed lowess normalization. Then linear model and empirical Bayes smoothing analyses were combined to obtain the statistics value and fold change of each spot. Significant candidates were selected with values of B>0. All of above calculations were done with the limma (http://bioinf.wehi.edu.au/limma/) package within the R environment (http://cran.r-project.org/).
|
| |
|
| Submission date |
Jan 20, 2010 |
| Last update date |
Jan 21, 2010 |
| Contact name |
Kunlin Zhang |
| Organization name |
Chinese Academy of Sciences
|
| Street address |
DaTun Road A4
|
| City |
Beijing |
| ZIP/Postal code |
100101 |
| Country |
China |
| |
|
| Platform ID |
GPL9952 |
| Series (1) |
| GSE19974 |
Systematic evaluation of genome-wide methylated DNA enrichment using a CpG island array |
|
