How does the Illumina direct hybridization assay differ from two colour arrays?
In “two colour” microarray experiments two samples are labeled with distinct fluorescent dyes, commonly Cy3 and Cy5, and combined prior to hybridization. The microarray is scanned to detect the fluorescent signals generated by each of the fluorescent dyes. For each immobilized probe sequence the ratio of signal intensities generated by each of the fluorescent dyes is an indication of the relative abundance of that transcript in each sample. Illumina’s Whole-Genome Gene Expression system uses a “direct hybridization” assay whereby biotin labeled samples are hybridized individually to an array. Following hybridization transcripts are detected using Cy3 conjugated streptavidin and scanned. The signal intensities generated give an indication of the absolute abundance of transcripts within that population. The signal intensities generated from separate arrays can be compared to give an indication of the differences between the two sample populations.
