10 protocols
AccessionType
 
normalization data transformation protocol
Data was background substracted and LOESS normalized within arrays and scale normalized between arrays using R (2.15)/Bioconductor/Limma
array scanning and feature extraction protocol
Slides were scanned using an Agilent DNA Microarray Scanner G2505C; scan software: Agilent Scan Control Version A.8.1.3; quantification software: Agilent Feature Extraction Version 10.5.1.1 (FE Protocol GE_105_Dec08).(Parameters: Scanning hardware = G2505A DNA microarray scanner [Agilent], Scanning software = Feature Extraction Software [Agilent])
nucleic acid hybridization to array protocol
Agilent Two-Color Microarray-Based Gene Expression (Hybridization)
Version: 5.7
Agilent publication number: G4140-90050
URL: http://www.chem.agilent.com/Library/usermanuals/Public/G4140-90050_Two-Color_GE_5.7.pdf
This document describes Agilent's recommended procedures for the preparation and labeling of complex biological targets and hybridization, washing, scanning, and feature extraction of Agilent 60-mer oligonucleotide microarrays for microarray-based two-color gene expression analysis.
nucleic acid labeling protocol
Agilent Two-Color Microarray-Based Gene Expression (Quick Amp Labeling)
Version: 5.7
Agilent publication number: G4140-90050
URL: http://www.chem.agilent.com/Library/usermanuals/Public/G4140-90050_Two-Color_GE_5.7.pdf
This document describes Agilent's recommended procedures for the preparation and labeling of complex biological targets and hybridization, washing, scanning, and feature extraction of Agilent 60-mer oligonucleotide microarrays for microarray-based two-color gene expression analysis.
pool
A equal aliquot of Cy3 labeled extracts from samples TAM 21, TAM 23, TAM 22, TAM 29 was pooled to build a reference.
nucleic acid extraction protocol
RNA was isolated using the Nucleospin RNA II kit (Macherey-Nagel, Dueren, Germany) according to manufacturers instructions.
growth protocol
Ascites was collected from patients with high grade serous ovarian carcinoma undergoing primary surgery at the University Hospital in Marburg. Informed consent was obtained from all patients according to the protocols approved by the local ethical committee. Peripheral blood mono-nuclear cells (PBMC) were obtained from healthy adult volunteers for monocyte-derived macrophage (MDM) stimulation. Mononuclear cells were isolated from ascites and peripheral blood by ficoll (GE Heathcare/PAA, Cölbe, Germany) density gradient centrifugation and further purified by magnetic cell sorting (MACS) using CD14 microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). Purified TAMs and MDMs were directly analyzed by FACS, lysed in PeqGold (Peqlab, Erlangen, Germany) for RNA preparation or cultured in serum-free macrophage medium (Mph medium; Life Technologies, Darmstadt, Germany).
treatment protocol
CD14+ monocytes and TAMs were cultured in serum-free macrophage medium (Mph medium; Life Technologies, Darmstadt, Germany). Monocyte-derived macrophages (MDMs) were differentiated from CD14+ monocytes of healthy volunteers for 5-7 d at 1x106 cells/ml in Mph medium supplemented with either MCSF or GMCSF or DMSO control (Immunotools, Friesoythe, Germany).
normalization data transformation protocol
Data was log2 transformed