|
| Status |
Public on Mar 10, 2016 |
| Title |
Tumor11 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
glioblastoma tissue
|
| Organism |
Homo sapiens |
| Characteristics |
clinical_subgroup: long-term survivor (LTS)
tissue type: glioblastoma
|
| Growth protocol |
Not applicable
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Tumor tissue was snap-frozen and stored at -80°C until further processing. DNA was extracted with the QIAmp DNA Mini Kit (Qiagen) from samples found eligible in terms of tumor cell content (>60%) and necrosis (<20%). Analyte concentration and quality were determined using the Nanodrop 2000 spectrophotometer (Thermo Scientific) and Bioanalyzer 2100 (Agilent). For enrichment of hypermethylated DNA fractions, Methyl-CpG-binding domain (MBD)-Fc protein was produced and MCIp performed as previously described (Schilling et al., Genomics 2007). Briefly, 75 µg of MBD-Fc protein was coupled to 50 µl SIMAG protein A magnetic beads (Chemicell) at 4°C overnight. After binding of 2 µg sonicated DNA, the DNA was eluted with increasing NaCl concentrations (300-1000 mM) using the MagnetoPure-Micro bead separator (Chemicell). Lowly and highly methylated DNA was eluted at the lowest and highest concentrations, respectively. DNA enrichment was monitored by real-time PCR targeting known unmethylated and methylated DNA regions.
|
| Label |
Cy5
|
| Label protocol |
Highly methylated glioblastoma and normal brain control DNA were labeled with Alexa Fluor 5 and Alexa Fluor 3, respectively.
|
| |
|
| Channel 2 |
| Source name |
normal brain tissue
|
| Organism |
Homo sapiens |
| Characteristics |
tissue type: normal brain
|
| Growth protocol |
Not applicable
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Tumor tissue was snap-frozen and stored at -80°C until further processing. DNA was extracted with the QIAmp DNA Mini Kit (Qiagen) from samples found eligible in terms of tumor cell content (>60%) and necrosis (<20%). Analyte concentration and quality were determined using the Nanodrop 2000 spectrophotometer (Thermo Scientific) and Bioanalyzer 2100 (Agilent). For enrichment of hypermethylated DNA fractions, Methyl-CpG-binding domain (MBD)-Fc protein was produced and MCIp performed as previously described (Schilling et al., Genomics 2007). Briefly, 75 µg of MBD-Fc protein was coupled to 50 µl SIMAG protein A magnetic beads (Chemicell) at 4°C overnight. After binding of 2 µg sonicated DNA, the DNA was eluted with increasing NaCl concentrations (300-1000 mM) using the MagnetoPure-Micro bead separator (Chemicell). Lowly and highly methylated DNA was eluted at the lowest and highest concentrations, respectively. DNA enrichment was monitored by real-time PCR targeting known unmethylated and methylated DNA regions.
|
| Label |
Cy3
|
| Label protocol |
Highly methylated glioblastoma and normal brain control DNA were labeled with Alexa Fluor 5 and Alexa Fluor 3, respectively.
|
| |
|
| |
| Hybridization protocol |
Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequential
|
| Scan protocol |
Scanned on an Agilent G2505B scanner.
Images were quantified using Agilent Feature Extraction Software.
|
| Description |
DNA was enriched for hypermethylated fractions by Methyl-CpG Immunoprecipitation (MCIp)
|
| Data processing |
Scanning raw data were processed using the statistical environment R. Background correction and log2 transformation were performed according to the NormExp method with offset = 50 (Ritchie et al., Bioinformatics 2007). Intensity-based LOESS normalization was applied to rank-invariant probes and negative controls in order to reduce technical noise between samples (Tseng et al., Nucleic Acids Res 2001). For intra-array normalization, log-intensity ratios (M-value) and log-intensity averages (A-values) were scaled to have the same median-absolute value across the array.
|
| |
|
| Submission date |
Nov 02, 2015 |
| Last update date |
Mar 10, 2016 |
| Contact name |
Christel Herold-Mende |
| Organization name |
Heidelberg University Hospital
|
| Department |
Department of Neurosurgery
|
| Lab |
Experimental Neurosurgery
|
| Street address |
Im Neuenheimer Feld 400
|
| City |
Heidelberg |
| ZIP/Postal code |
69117 |
| Country |
Germany |
| |
|
| Platform ID |
GPL9767 |
| Series (1) |
| GSE74561 |
LOC283731 promoter hypermethylation prognosticates survival after radiochemotherapy in IDH1 wild-type glioblastoma patients |
|
