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The Illumina RNA microarray technology is called BeadArray and it is a fluorescence-based assay to evaluate gene expression by messenger RNA (mRNA) level. A BeadArray is created by synthesizing single-stranded 50 nucleotide DNA segments (probes) and then immobilizing them on thousands of individual microscopic beads. Each bead is coated in one probe and each probe represents a single gene. The beads are assembled in microwells on fiber optic bundles or silica slides to make a BeadChip. Commercial BeadChips include the HumanHT-12 v4.0 for genome-wide coverage of well characterized genes and MouseRef-8 v2.0 for the mouse whole genome. To create the target sequences for a one-color assay, mRNA is purified from the sample to be analyzed, reverse transcribed, and then amplified by in vitro transcription (IVT) into single-stranded antisense RNA (aRNA). Biotin labeled nucleotides are incorporated during IVT and the labeled aRNA is digested. These target sequences are then hybridized to the microarray during which they bind the complementary single-stranded probes. The unbound targets are then washed away and a Cy3-labeled biotin antibody is added. The microarray is laser scanned by an Illumina-compatible instrument, such as a HiScan or iScan, to measure the fluorescent emission, which is correlated to the level of transcription for each gene with Illumina-compatible software. The applications of RNA microarrays include the study of gene regulation, cell fate determination, the identification of disease-related genes, analysis of the effects of drugs on gene expression, and tumor typing for personalized cancer treatments. (Credit: Brooke Anderson-White)
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