DNA/RNA Analysis:

• Nanodrop ND-1000. The NanoDrop ND-1000 UV-Vis scanning spectrophotometer enables accurate analyses of 1-2ul samples over a large spectrum (220-750nm). The measurement is made, typically in less than 10 seconds, and the spectrum and its analysis is shown on the screen of the attached PC and archived on the PC. The small volume requirements of this instrument are critical for quantitation of the small samples frequently obtained from clinical specimens.
• Agilent 2100 Bioanalyzer. The Agilent 2100 Bioanalyzer uses microfluidics technology to provide electrophoresis analysis of DNA, RNA, proteins and cells. The primary use in the Core lab is for determination of total RNA quality for samples prepared for microarray-based gene expression analysis. The key advantage of this instrument is that it allows analysis of total RNA samples containing as little as 25 picograms. This is critical for samples derived from needle core biopsies where RNA yields are too small to allow traditional gel-based analysis methods. Up to12 samples can be processed in 30 minutes.

Genotyping:

• LightTyper (Roche Applied Sciences). We have recently acquired a new instrument for genotyping SNPs that utilizes melting curve analysis in a 96-well format. This assay is based on real time detection of probe melting temperature difference between perfect match and single base mismatches of the labeled probe and the amplicon containing the target polymorphism. Melting temperatures are determined through the detection of changes in probe fluorescence that occurs upon melting as the samples are heated. This assay has lower reagent costs and requires substantially less labor than the GBA assay (see below) we have been using for most of our SNP genotyping assays. We are currently in the process of converting all of our SNP assays to the LightTyper format.
• Genetic Bit Analysis. Genetic Bit Analysis (GBA or minisequencing) is a solid-phase primer extension-based assay for genotyping SNPs that has been used in the Core lab to run the majority of genotyping assays. We have recently begun to use the LightTyper system described above to replace the GBA method due to its lower reagent and labor costs. GBA will remain an option for analysis of SNPs that are resistant to genotyping using the LightTyper method.
• GeneChip Mapping 10K Array (Affymetrix). The recently introduced GeneChip Mapping 10K Array has the ability to generate over 10,000 genotypes on a single array with an average resolution of one SNP every 210kb on the genome. This assay requires 250ng of DNA for each sample and provides automated genotype calling with extensive annotation and links to public databases such as the UCSC Genome Browser. We are currently evaluating this technology in the Core Lab and anticipate that we will make this available Investigators for gene mapping studies within the next few months.

Gene Expression:

• Affymetrix GeneChip Technology. Affymetrix GeneChips remain the principle tool for high throughput gene expression analysis. There are several advantages that make this technology highly suitable for application to clinical studies. These include comprehensive transcriptome analysis on a single array (over 47,000 transcripts on the human 133 2.0 Plus array), a steady drop in cost/transcript from $0.33/gene in 2000 to $0.0096/gene in 2004, and highly standardized array manufacturing and sample processing protocols making analysis of data from different institutes or facilities possible. While we anticipate the continued use of GeneChips as a primary tool for gene expression analysis, we will continue to evaluate alternative technologies that may be more suitable or cost effective for specific applications. These include smaller focused arrays for screening large numbers of samples for expression patterns of a defined set of genes as well as non-array technologies.

Data/Statistical Analysis:

• One critical component of the DNA Array Core Facility is the expertise in the use of software tools for gene expression data/statistical analysis. The Core offers access to computer workstations and training and assistance in the use of several software packages specifically designed for analysis of microarray data. These include both commercial products (GeneSpring, Silicon Genetics; ImaGene, Biodiscovery; GCOS, Affymetrix) and publicly available softwares (BRB ArrayTools, NCI; SAM, Stanford; RMA, UC Berkeley; dChip, Harvard). These software products are used for image analysis, statistical testing, class prediction, clustering and estimation of expression levels from processed image files.

For more information about services offered, please call the Core Facility at (858) 784-2263