A reverse transcription with indirect labeling was performed using 10 ug of total RNA. The internal standard mixture was used for quantification/ normalization and estimation of experimental variation [de Longueville F, et al. Biochem. Pharmacol. 64, 137-49, 2002; Chen & Bittner. J. Biomedical Optics 2, 364-374, 1997]. Each DualChip(R) consists of two identical microarrays on the same slide, with two separate hybridization frames. This DualChip®-concept enables two complete gene expression experiments to be run on the same slide, using a one-color labeling technique.
Hybridization protocol
The DualChip(R) hybridization was carried out overnight (16 hours) at 60°C according to the DualChip(R) instruction manual.
Scan protocol
Eppendorf platform is an open system. Different scanner technologies and settings have been used by the different test sites.
Description
MAQC sample
Data processing
Each array was scanned at three different PMT gain settings. Eppendorf has developed specialized data analysis scheme treating the scanning, the background correction and normalization in a suited framework for low density arrays generating array to array gene expression ratio. The data sets submitted are divided in two groups. The first data set provides gene expression ratio comparing two samples and constitutes the standard result for the Eppendorf platform.
If the signal intensity for both the experiment and the reference are acceptable a ratio is termed "quantitative". If only one intensity is acceptable (experiment or reference), the ratio is said "qualitative". If both intensities are not acceptable, the ratio is called "not in linear range".
