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The Affymetrix RNA microarray technology is called GeneChip and it is an in situ probe generation platform with a fluorescence-based assay to evaluate gene expression by messenger RNA (mRNA) level. A GeneChip is created by synthesizing single-stranded 25 nucleotide DNA segments (probes) on a glass slide by photolithography masking. Each probe represents a single gene. Commercial GeneChips include genome-wide and custom Human SNP Arrays and RNA arrays for several species. 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 fluorescent streptavidin-phycoerythrin is added to bind the biotin. The microarray is laser scanned by an Affymetrix-compatible instrument to measure the fluorescent emission, which is correlated to the level of transcription for each gene with Affymetrix-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. The first GeneChip was for HIV genotyping. (Credit: Brooke Anderson-White)
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