normalization data transformation protocol
the signal intensities from single experiment raw data lists are normalized by dividing the intensities values by their median ID_REF = VALUE = Normalized signal intensity
array scanning protocol
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent's Microarray Scanner System (Agilent Technologies). The Agilent Feature Extraction Software (FES) v9,1 was used to read out and process the microarray image files. The software determines feature intensities including background subtraction.
The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using Agilent Gene Expression Hybridization Kit (Agilent Technologies). 1,65µg Cy3-labeled fragmented cRNA in hybridization buffer was hybridized overnight (17 hours, 65°C) to Agilent Whole Human Genome Oligo Microarrays 4x44k using Agilent's recommended hybridization chamber.
1µg of total RNA sample was used for linear T7-based amplification. To produce Cy3-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Quick Amp Labeling Kit/Low RNA Input Linear Amp Kit (Agilent Technologies) following manufacturer's protocol.
Mononuclear cells from human bone marrow were separate were separate by Ficoll and cultivated in medium alphaMEM with 10% of FCS and induced to differentiate in osteoblastic lineage during 21 days
sample treatment protocol
Bone marrow mesenchymal stromal cells induced to differentiate in osteoblastic lineage during 21 days
nucleic acid extraction protocol
RNA was isolated using standard RNA extraction protocols (NucleoSpin RNA II, Macherey-Nagel). RNA samples were quality-checked via the Agilent 2100 Bioanalyzer Platform (Agilent Technologies)