|
| Status |
Public on Jun 01, 2013 |
| Title |
PANC1 Non-invasive (Top) [Chip 1] |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Pancreatic Cancer Cell Line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: PANC1
phenotype: Non-invasive
dna type: Non-methylated Genomic DNA
|
| Treatment protocol |
70,000-100,000 cells were seeded in RPMI with nothing else in 24 well matrigel chambers.Cells were migrated toward stem cell media (SCM) for 24 hours containing DMEM/F12 with human supplementation of 10 ng/mL bFGF, 20 ng/mL EGF, 5 µg/mL insulin, 5 µg/mL transferrin and 5ng/ml sodium selenite along with 0.4% BSA
|
| Growth protocol |
PANC1 and HPAC cell lines were grown according to methods from ATCC.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For the isolation of DNA from both non-invasive and invasive cells the DNeasy kit from Qiagen (Valencia, CA) was used and parallel invasion chambers were setup. For non-invading cells, the bottom of the membrane was scrubbed with a cotton swab and cells on top were trypsinized and harvested in 200 µL of PBS followed by the direct addition of lysis buffer or stored at -80ºC. For bottom ‘invading cells’ the top of the membrane was scrubbed with a cotton swab and the membrane was removed and placed directly into lysis buffer or stored at -80ºC until needed. MethylCollector (Active Motif, Carlsbard, CA) assay was used to isolate methylated and non-methylated fractions of DNA. The kit utilizes histidine-tagged MeBP2 (methyl-binding protein 2) and magnetic bead separation.
|
| Label |
Cy3
|
| Label protocol |
The isolated methylated and non-methylated DNA from each sample (as little as 5 ng) was then amplified in a series of PCR reactions following the mammalian ChIP-on-chip protocol from Agilent. The input DNA was labeled with Cy3-dUTP and the methylated DNA with Cy5-dUTP.
|
| |
|
| Channel 2 |
| Source name |
Pancreatic Cancer Cell Line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: PANC1
phenotype: Non-invasive
dna type: Methylated Genomic DNA
|
| Treatment protocol |
70,000-100,000 cells were seeded in RPMI with nothing else in 24 well matrigel chambers.Cells were migrated toward stem cell media (SCM) for 24 hours containing DMEM/F12 with human supplementation of 10 ng/mL bFGF, 20 ng/mL EGF, 5 µg/mL insulin, 5 µg/mL transferrin and 5ng/ml sodium selenite along with 0.4% BSA
|
| Growth protocol |
PANC1 and HPAC cell lines were grown according to methods from ATCC.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For the isolation of DNA from both non-invasive and invasive cells the DNeasy kit from Qiagen (Valencia, CA) was used and parallel invasion chambers were setup. For non-invading cells, the bottom of the membrane was scrubbed with a cotton swab and cells on top were trypsinized and harvested in 200 µL of PBS followed by the direct addition of lysis buffer or stored at -80ºC. For bottom ‘invading cells’ the top of the membrane was scrubbed with a cotton swab and the membrane was removed and placed directly into lysis buffer or stored at -80ºC until needed. MethylCollector (Active Motif, Carlsbard, CA) assay was used to isolate methylated and non-methylated fractions of DNA. The kit utilizes histidine-tagged MeBP2 (methyl-binding protein 2) and magnetic bead separation.
|
| Label |
Cy5
|
| Label protocol |
The isolated methylated and non-methylated DNA from each sample (as little as 5 ng) was then amplified in a series of PCR reactions following the mammalian ChIP-on-chip protocol from Agilent. The input DNA was labeled with Cy3-dUTP and the methylated DNA with Cy5-dUTP.
|
| |
|
| |
| Hybridization protocol |
Samples were applied to Agilent’s 2x244K Human Promoter Tiling Arrays for 40 hours at 65ºC and washed for 5 minutes in wash buffer 1 and then 5 minutes in wash buffer 2.
|
| Scan protocol |
The arrays were scanned using a Gene Pix 4000B scanner (Molecular Devices, Sunnyvale, CA) with GenePix Pro software version 6.1 and extracted using Agilent’s Feature Extraction software version 9.5.3.1.
|
| Description |
Input verses methyl collector
|
| Data processing |
The data was annotated using Agilent’s ChIP Analytics software version 4.0. Normalization was carried out using a blank subtraction model and statistical stringency (p-value) between 0.01-0.05 was applied using a Whitehead Per-Array Neighbourhood Analysis. This analysis allowed for the determination of differentially methylated genes between non-invasive and invasive cells.
Methylated probes appear in the array with a value of 1 in the ISINBOUND Region and un-methylated probes array with a value of 0. Using FileMaker Pro Software (version 9) the full text files were imported and the find option was used in order to query probes that were methylated in the Non-Invasive cells and non-methylated in the Invasive cells.
|
| |
|
| Submission date |
May 21, 2012 |
| Last update date |
Jun 01, 2013 |
| Contact name |
Lei Sun |
| E-mail(s) |
lei.sun@nih.gov
|
| Phone |
301-846-6867
|
| Fax |
301-846-6104
|
| Organization name |
Frederick National Laboratory for Cancer Research
|
| Department |
Laboratory of Cancer Prevention
|
| Lab |
Cancer Stem Cell Section
|
| Street address |
1050 Boyles Street
|
| City |
Frederick |
| State/province |
MD |
| ZIP/Postal code |
21702 |
| Country |
USA |
| |
|
| Platform ID |
GPL4124 |
| Series (1) |
| GSE38076 |
CpG Methylation profile of Invasive Pancreatic Cancer Cells compared to Non-invasive |
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