Digoxigenin-UTP labeled cRNA was generated from 1 µg of total RNA for each MAQC sample (A, B, C, D) using Applied Biosystems NanoAmp RT-IVT Labeling Kit (P/N 4365715) according to the manufacturer’s protocol.
Hybridization protocol
Array hybridization, array processing, chemiluminescence detection, image acquisition, and analysis were performed using Applied Biosystems Chemiluminescence Detection Kit and Applied Biosystems 1700 Chemiluminescent Microarray Analyzer following manufacturer’s protocol.
The Expression Array System Software suite performs the auto-gridding, feature extraction, fluorescence normalization, and signal data generation. The quantification data contain many distinct measurements for each probe, including the three basic measurements: Signal, S/N, and Flag values. The Signal value is the fully corrected, background subtracted measurement of chemiluminescent signal for gene expression values. The S/N value represents the ratio of signal above noise, or the measurement uncertainty of the probe signal, and can be used as a confidence level for the probe measurement. An S/N of 3 represents approximately a 99.95% confidence that the probe is detected above the background noise. In situations where the probe showed S/N < 1, the signal measurement is replaced with a 1 SDEV upper limit based on its probe signal SDEV.
The signal value is the fully corrected, background subtracted measurement of chemiluminescent signal for gene expression values. Data were quantile-normalized per test site (20 arrays).
Flag_Detection
An S/N of 3 represents approximately a 99.95% confidence that the probe is detected above the background noise.
