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Sample GSM620743 Query DataSets for GSM620743
Status Public on Nov 01, 2012
Title Colorectal carcinoma CRC15 [MCIp]
Sample type genomic
 
Channel 1
Source name colorectal carcinoma
Organism Homo sapiens
Characteristics gender: male
age: 69
crc location: Sigma-Ca
ms status: microsatellite stable (MSS)
kras- and braf mutational status: KRAS-mut; 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

Data table header descriptions
ID_REF
LOG10_CHANNEL_R Log10 transformed, normalized signal intensity of cancer sample
LOG10_CHANNEL_G Log10 transformed, normalized signal intensity of normal sample
VALUE Linear normalized log10 process red (cancer) versus green (normal) signal
AVE_LOG10 Average Log10 signal intensity of both channels

Data table
ID_REF LOG10_CHANNEL_R LOG10_CHANNEL_G VALUE AVE_LOG10
189370 2.162062019 2.236056563 -0.073994544 2.199059291
113763 2.238981464 2.716954431 -0.477972967 2.477967948
1449 2.715172295 2.687140722 0.028031573 2.701156508
159414 3.504634491 3.569756632 -0.065122141 3.537195561
176668 1.704790501 2.222180965 -0.517390465 1.963485733
19052 3.37574827 3.496717377 -0.120969107 3.436232824
172902 2.56916233 2.743493959 -0.174331629 2.656328144
142718 3.279779837 3.947868627 -0.668088790 3.613824232
130519 3.757994593 3.500870508 0.257124085 3.629432551
194646 3.473154005 3.397586297 0.075567708 3.435370151
203686 2.697723791 3.278370168 -0.580646377 2.988046979
60442 2.069182481 2.249057025 -0.179874544 2.159119753
25695 3.143147255 2.961820614 0.181326641 3.052483935
91503 2.895237296 3.253482898 -0.358245602 3.074360097
183659 2.642720174 3.042337213 -0.399617039 2.842528694
148314 3.274007899 3.95889146 -0.684883560 3.61644968
119278 3.017771847 3.883109794 -0.865337947 3.45044082
42655 3.21007816 3.875572593 -0.665494433 3.542825376
151395 3.149799324 3.901808828 -0.752009504 3.525804076
69693 3.037224046 3.86260357 -0.825379524 3.449913808

Total number of rows: 237202

Table truncated, full table size 12692 Kbytes.




Supplementary file Size Download File type/resource
GSM620743_CRC15_CGH-v4_95_Feb07.txt.gz 67.8 Mb (ftp)(http) TXT
Processed data included within Sample table

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