normalization data transformation protocol
The Agilent Feature Extraction Software (FES 10.5.1.1 ) was used to read out and process the microarray image files. ID_REF = VALUE = median normalized signal intensities
array scanning protocol
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (G2505C, Agilent Technologies, Palo Alto, USA).
Cy3 labeled cRNAs were hybridized overnight (17 hours, 65°C) to an Agilent Whole Human Genome Oligo Microarrays (44K) using Agilent’s recommended hybridization chamber and oven.Finally, microarrays were washed once with wash buffer 1 for 1 min at RT followed by a second wash with wash buffer 2 for 1 min at 37°C and a final wash step with acetonitrile for 30 sec at RT.
1 µg of each RNA was used as template to produce Cy3- labeled cRNA. The RNA samples were amplified and labeled using the Agilent Quick Amp Labeling Kit (Agilent Technologies) following the manufacturer’s protocol.
nucleic acid extraction protocol
RNA was prepared using standard RNA extraction protocols (Trizol). Quality was checked via the Agilent 2100 Bioanalyzer platform.
sample treatment protocol
Human immortalized mesenchymal stromal cells (SCP-1) were incubated with conditioned medium of the breast carcinoma cell line MCF-7 for 24, 48, and 72 hours at 37 ºC in a humidified incubator with 5% CO2.