disease state: osteosarcoma origin: Met age: 18 gender: Male developed metastases: Yes
Extracted molecule
total RNA
Extraction protocol
RNA was extracted by TRIzol (OS3, 4, 12, 18, 21, 41, 47, 48, 50, 51, 53, 55) or GTC (OS11, 14, 15, 17, 19, 20, 23, 25, 29, 30 and 32) using standard protocols.
Label
Digoxigenin
Label protocol
A 1-μg of total RNA was amplified and converted to digoxigenin-labeled cRNA by using the NanoAmp RT-IVT Labeling kit (Applied Biosystems) in accordance with the manufacturer's protocol.
Hybridization protocol
The Chemiluminescent Detection kit (Applied Biosystems) was used for the preparation of labeled cRNA, and hybridization and washing solutions. Both digoxigenin-UTP and the anti-digoxigenin -AP were provided by Roche Diagnostics (Mannheim, Germany). Digoxigenin-labeled probes were hybridized to the microarrays overnight (16 h) at 55 °C.
Scan protocol
Chemiluminescence signals were detected by using AB1700 Chemiluminescent Microarray Analyzer (Applied Biosystems). Image preparation, signal intensity quantification and initial analysis were performed by using the accompanying software from Applied Biosystems.
Data processing
The data were further processed with the R package ABarray, which is part of the Bioconductor project (http://www.R-project.org). The data were quantile normalized, log2 transformed and missing values were imputed using average values from the other arrays in the subgroup. The osteosarcoma samples were divided into two groups of primary or metastatic origin. Weakly expressed probes were filtered away by defining that a probe is only detected if it has a signal-to-noise ratio (SNR) ≥ 3 in at least 50% of the samples in either subgroup primary or metastatic.
