|
| Status |
Public on Nov 01, 2012 |
| Title |
Colorectal carcinoma CRC13 [MCIp] |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
colorectal carcinoma
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
age: 68
crc location: Sigma-Ca
ms status: microsatellite stable (MSS)
kras- and braf mutational status: KRAS-wt; BRAF-wt
|
| Growth protocol |
Colorectal cancer samples were collected from 9 patients who underwent colon resection for biopsy-proven invasive colorectal adenocarcinoma at the University of Regensburg. The study was performed in agreement with the Institutional ethical review board of the University of Regensburg (05/003). DNA from frozen colon tissues was isolated using the PUREGENE™ DNA Purification Kit (Gentra, Minneapolis, USA) according to the supplier’s recommendation. Three normal colon DNAs (male, age 50-63) were purchased from BioChain Institute Inc. (Hayward, CA)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
MCIp enrichment of methylated DNA was performed essentially as described (Gebhard et al. 2006, Cancer Research). In brief, genomic DNA was sonicated to a mean fragment size of 350-400 bp. Two µg of each sample were incubated with 150 µl Protein A-Sepharose 4 Fast Flow beads (GE Healthcare) coated with 60 µg purified MBD-Fc protein in 2 ml Ultrafree-MC centrifugal filter devices (Amicon/Millipore) for 3 h at 4°C in buffer containing 300 mM NaCl. Beads were centrifuged to recover unbound DNA fragments (300 mM fraction) and subsequently washed with buffers containing increasing NaCl concentrations (400, 500, 550 mM). Densely CpG-methylated DNA was eluted with a high-salt buffer (1000 mM NaCl) and all fractions were desalted using the MinElute PCR purification kit (Qiagen). The separation of CpG methylation densities of individual MCIp fractions was controlled by qPCR using primers covering the imprinted SNRPN
|
| Label |
Alexa Fluor 5–dCTP
|
| Label protocol |
Enriched methylated DNA fragments of the high-salt MCIp fractions were labeled with Alexa Fluor 5–dCTP (cancer cells) and Alexa Fluor 3–dCTP (normal cells) using the BioPrime Total Genomic Labeling System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
|
| |
|
| Channel 2 |
| Source name |
normal colon
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
sample: normal colon; mixture of three samples (age 50-63)
|
| Growth protocol |
Colorectal cancer samples were collected from 9 patients who underwent colon resection for biopsy-proven invasive colorectal adenocarcinoma at the University of Regensburg. The study was performed in agreement with the Institutional ethical review board of the University of Regensburg (05/003). DNA from frozen colon tissues was isolated using the PUREGENE™ DNA Purification Kit (Gentra, Minneapolis, USA) according to the supplier’s recommendation. Three normal colon DNAs (male, age 50-63) were purchased from BioChain Institute Inc. (Hayward, CA)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
MCIp enrichment of methylated DNA was performed essentially as described (Gebhard et al. 2006, Cancer Research). In brief, genomic DNA was sonicated to a mean fragment size of 350-400 bp. Two µg of each sample were incubated with 150 µl Protein A-Sepharose 4 Fast Flow beads (GE Healthcare) coated with 60 µg purified MBD-Fc protein in 2 ml Ultrafree-MC centrifugal filter devices (Amicon/Millipore) for 3 h at 4°C in buffer containing 300 mM NaCl. Beads were centrifuged to recover unbound DNA fragments (300 mM fraction) and subsequently washed with buffers containing increasing NaCl concentrations (400, 500, 550 mM). Densely CpG-methylated DNA was eluted with a high-salt buffer (1000 mM NaCl) and all fractions were desalted using the MinElute PCR purification kit (Qiagen). The separation of CpG methylation densities of individual MCIp fractions was controlled by qPCR using primers covering the imprinted SNRPN
|
| Label |
Alexa Fluor 3–dCTP
|
| Label protocol |
Enriched methylated DNA fragments of the high-salt MCIp fractions were labeled with Alexa Fluor 5–dCTP (cancer cells) and Alexa Fluor 3–dCTP (normal cells) using the BioPrime Total Genomic Labeling System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
|
| |
|
| |
| Hybridization protocol |
Comparative MCIp hybridizations on CpG island oligonucleotide microarrays (Agilent) were performed using the recommended, stringent protocol (Agilent).
|
| Scan protocol |
Scanned on an Agilent scanner at 5 µm resolution.
Images were analysed and data extracted using Agilent Feature Extraction Software (version 9.5.1).
|
| Description |
Comparative MCIp-on-Chip for colorectal carcinoma and normal human colon
|
| Data processing |
Linear normalized, background subtracted VALUE data were obtained from log10 transformed, processed Red signal/processed Green signal values derived from image analysis with Agilent Feature Extraction Software (version 9.5.1). Processed signal intensities were further normalized using GC-dependent regression and imported into Microsoft Office Excel 2007 for further analysis.
|
| |
|
| Submission date |
Nov 09, 2010 |
| Last update date |
Nov 01, 2012 |
| Contact name |
Michael Rehli |
| E-mail(s) |
michael.rehli@klinik.uni-r.de
|
| Organization name |
University Hospital Regensburg
|
| Department |
Internal Med III
|
| Street address |
F.-J.-Strauss-Allee 11
|
| City |
Regensburg |
| ZIP/Postal code |
93042 |
| Country |
Germany |
| |
|
| Platform ID |
GPL8544 |
| Series (2) |
| GSE25221 |
CpG island methylation in colorectal cancer |
| GSE25229 |
Genome-wide analysis of unbalanced chromosomal changes in colorectal carcinoma |
|
