The Core Lab provides gene expression profiling and DNA genotyping services using prefabricated chips by Affymetrix. Chips have the advantage of assessing effects of treatment/conditions on tens of thousands of genes simultaneously, identifying target gene for further investigation. The Affymetrix GeneChip provides consistent and easily comparable results.
GeneChip microarrays consist of small DNA fragments, referred as probes, chemically synthesized at specific locations on a coated quartz surface. The precise location where each probe is synthesized is called a feature, and millions of features can be contained on one array. By extracting and labeling nucleic acids from experimental samples, and then hybridizing those prepared samples to the array, the amount of label can be monitored at each feature, enabling a wide range of applications on a whole genome scale including gene- and exon-level expression analysis, novel transcript discovery, genotyping, and resequencing. (Affymetrix Tech-Note)
Expression and Genotyping
Investigators study gene expression by measuring the number of RNA copies a gene produces. The gene expression microarray is a tool that indicates how much RNA each gene is making. A typical Afffymetrix gene expression array experiment involves the following steps:
- Preparation of fluorescent labeled target from mRNA.
- Hybridization of the labeled target to the microarray.
- Washing, staining, and scanning of the array.
- Analysis of the scanned image and generation of gene expression profiles.
There are only four molecules, or "bases", in every DNA chain: (A), (G), (T), (C). These four molecules partner: C partners with G and T partners with A. Affymetrix DNA arrays use this base pairing attraction hybridization to help researchers identify each SNP genotype.
Requirements for Submitting Samples
Quality of the RNA is the single most important determinant of a successful GeneChip analysis assay. Particularly, differential degradation of RNA can lead to erroneous conclusions. We recommend TRIzol reagent for isolation of total RNA from tissue specimens as well as cultured and blood cells. The A260/A280 ratio should be at least 1.6 for pure RNA. The gel profile should exhibit a 28S band that is 2 times more intense than 18S ribosomal RNA and no degradation.
- Total RNA from using TRIzol isolation: minimum 15μg in 30μl of RNase free water or a visible pellet in 75% ethanol.
- Total RNA from using QIAshedder and RNeasy: minimum 10μg in 11μl of RNase free
- Poly(A)+ mRNA from using Oligotex: minimum 3μg in 6μl of RNase free water.