type: osteoblast growth protocol: We used human osteoblasts derived from osteogenic differentiated bone-marrow-derived mesenchymal stem cells as described previously (Cleton-Jansen AM, Anninga JK, Briaire-de Bruijn IH, Romeo S, Oosting J, Egeler RM, Gelderblom H, et al. Profiling of high-grade central osteosarcoma and its putative progenitor cells identifies tumourigenic pathways. Br J Cancer. 2009 Dec 15;101(12):2064 (PMID: 19997114)).
Extracted molecule
total RNA
Extraction protocol
RNA isolation, cDNA synthesis, cRNA amplification, Illumina Human-6 v2.0 Expression BeadChip hybridization, qPCR validation, and microarray data analysis were performed as previously described (Buddingh EP, Kuijjer ML, Duim RA, Burger H, Agelopoulos K, Myklebost O, et al. Tumor-infiltrating macrophages are associated with metastasis suppression in high-grade osteosarcoma: a rationale for treatment with macrophage-activating agents. Clin Cancer Res 2011 Apr 15;17(8):2110-2119).
Label
biotin
Label protocol
As per manufacturer's instructions.
Hybridization protocol
As per manufacturer's instructions.
Scan protocol
As per manufacturer's instructions.
Description
Gene expression data were exported from BeadStudio version 3.1.3.0 (Illumina) in GeneSpring probe profile format. Two supplementary/raw data files are present because each array that represents one Sample consists of 2 chips. The supplementary/raw data files represent the bead level data (2 raw .txt files per sample, containing all intensities per bead).
Data processing
Data were analyzed using the statistical language R. As Illumina identifiers are not stable and consistent between different chip versions, raw oligonucleotide sequences were converted to nuIDs. Data were transformed using the variance stabilizing transformation (vst) algorithm and subsequently normalized using robust spline normalization. Conversion to nuIDs, variance stabilizing transformation, and normalization of the data were all carried out using the Bioconductor package lumi. Quality control was performed using the Bioconductor package arrayQualityMetrics. Poor quality arrays were removed from further analyses.
