|
| Status |
Public on Aug 31, 2008 |
| Title |
CAD_replicate93 |
| Sample type |
RNA |
| |
|
| Source name |
Human monocytes from individuals with coronary artery disease
|
| Organism |
Homo sapiens |
| Characteristics |
Gender:men
Age:66
Collateral flow index: 0.2958
CAD_category:CAD
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Monocytes were separated from whole blood immediately after completion of the invasive study protocol. Following an erythrocyte lysis step (erythrocyte lysis buffer, Qiagen, Hilden, Germany), peripheral blood monocytes were isolated from leukocytes by the magnetic activated cell separation bead selection method (MACS, CD14 antibody linked to paramagnetic beads; Dynal AG, Oslo, Norway). RNA from monocytes was extracted immediately after their isolation using a membrane based system (RNeasy mini, Qiagen)
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 1 ug total RNA
|
| |
|
| Hybridization protocol |
Gene expression was analyzed using Affymetrix HG-U133A 2.0 arrays. : Starting with 1µg of total RNA, complementary RNA (cRNA) was generated. Double stranded complementary DNA (cDNA) was synthesized in two steps according to the manufacturer's protocol (Superscript ds cDNA Synthesis Kit, Invitrogen). cDNA cleanup was performed by phenol chloroform extraction. Biotinylated cRNA was synthesized by in vitro transcription using T7 polymerase (GeneChip Expression 3'-Amplification IVT labeling kit; Affymetrix). The resulting cRNA was purified using RNeasy kit (Qiagen), quantitated and quality was assessed before fragmentation at 94°C for 35 min. A total of 7 g cRNA was hybridized to the Affymetrix U133A 2.0 gene chip for 16h at 45°C. Washing, staining and scanning procedures were carried out according to the protocol of the manufacturer.
|
| Scan protocol |
The GeneChips were processed with a Affymetrix GeneChip® Scanner 3000 7G (Affymetrix) by using the current default settings. DAT image files of the microarrays were generated using GeneChip Operating Software (GCOS 1.4; Affymetrix).
|
| Description |
Gene expression data from an individual with coronary artery disease
|
| Data processing |
Microarray quality was assessed with the R software, in particular with the affyPLM package in Bioconductor 12 and its model based quality assessment methods 13. For each batch of microarrays the following procedures were performed: First, homogeneity of the probe intensity distributions was checked across arrays in a batch. Chips with aberrant distributions were marked for exclusion. Second, the probe level intensities for each Affymetrix probe set were fitted with robust weighted regressions in which the intensities are explained by a linear model with gene chip effects and probe effects. If the probes of a probe set behave consistently, the residuals of the fit are small. Outlier probe intensities result in larger absolute residuals and low weights. These are symptomatic for intensity measurements from regions of a gene chip that provide low quality data. Chips with extended areas of large absolute residuals were marked for exclusion. Third, we used the normalized un-scaled standard errors (NUSE) of the probe effects on each array. We found that excluding the arrays with a median NUSE of 1.05 or more was a good criterion summarizing the observations from all three procedures and so these arrays were excluded from further statistical analyses
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| |
|
| Submission date |
Apr 26, 2007 |
| Last update date |
Apr 08, 2009 |
| Contact name |
Pascal Meier |
| E-mail(s) |
pascalmeier@gmx.ch
|
| Phone |
+41316322111
|
| Fax |
+41316324560
|
| Organization name |
University Hospital
|
| Department |
Cardîology
|
| Street address |
Freiburgstrasse
|
| City |
Bern |
| ZIP/Postal code |
3010 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL571 |
| Series (1) |
| GSE7638 |
Expression data from monocytes of individuals with different collateral flow index CFI |
|
