Array Data Analysis

The images are analyzed using the Beadstudio software. Quality control and data analysis are carried out according to the instructions provided by Illumina. The quality control parameters are as follows:

Housekeeping Controls

The intactness of the biological specimen can be monitored by the housekeeping gene controls. These controls consist of probes to housekeeping genes, two probes per gene, which should be expressed in any cellular sample. The housekeeping values for all the arrays in one experiment should be all very similar, otherwise comparison data might not be accurate.

Hybridization Controls

Three types of controls comprise this category: Cy3-labeled Hyb Control, Low Stringency Hyb Control, and High Stringency Hyb Control. The Cy3-Labeled Hyb control consists of six probes with corresponding Cy3-labeled oligonucleotides present in the controls added to the cRNA before hybridization. Following successful hybridization, they produce a signal independent of both the cellular RNA quality and success of the sample prep reactions. Target oligonucleotides for the Cy3 Hyb controls are present at three concentrations (low, medium, and high), yielding gradient hybridization. As with the housekeeping controls, the values for the low, medium and high concentrations for all arrays in one experiment should be very similar. The Low Stringency Hyb Control contains four probes, corresponding to the medium- and high-concentration Cy3 Hyb control targets. In this case, each probe has two mismatch bases distributed in its sequence. If stringency is adequate, these controls yield very low signal. If stringency is too low, they yield signal approaching that of their perfect match counterparts in the Cy3 Hyb control category.

The High Stringency Hyb Control consists of one probe corresponding to a Cy3-labeled oligonucleotide target. The probe/target sequences have a very high G+C content, and should thus hybridize even if hybridization stringency is too high. Hybridizations with too-high stringency are detected on the basis of a signal present from this control in the absence of signal from the other hybridization control probes.

Negative Controls

This category consists of probes of random sequence selected to have no corresponding targets in the genomes. The mean signal of these probes defines the system background. This is a comprehensive measurement of background, representing the imaging system background as well as any signal resulting from non-specific binding of dye or cross-hybridization. The BeadStudio application uses the signals and signal standard deviation of these probes to establish gene expression detection limits.