1. Sample Preparation : Although the Core does not routinely process tissue samples for DNA and/or RNA, in selected cases Dr. Triche’s lab will accept freshly procured tissues, frozen tissue, tissue cultures, buccal smears, etc., and extract either RNA or DNA (or both, if sufficient material is submitted). This service is generally offered only to new investigators who seek to develop the necessary skills to perform whole-genome studies and who have no prior experience. The goal of this service is to teach such users or their lab staffs the basic principles of RNA or DNA extraction that will result in high quality material suitable for microarray analysis. Because this is not a standard service, potential users will need to contact Dr. Triche before submitting such specimens.
2. Gene Expression Profiling : Transcriptome profiling is offered for any species for which Affymetrix Gene Chips are available. In addition, custom arrays can be ordered by the user directly from Affymetrix for shipment to the Core. In each case, the user is responsible for submitting a sample for analysis in the form of total RNA. The Core will then analyze the amount, purity, and relative degradation. If there is sufficient RNA of adequate quality, the Core will prepare labeled cRNA for hybridization to the requested array. If initial QC suggests that the RNA is of inadequate quality, a test array will be run. If the results warrant full analysis, the same hybridization mix will be hybridized to the requested array. If insufficient RNA is present in the initial sample or after creating labeled cRNA, the user will be notified and no further action will be taken.
3. High Density SNP analysis : DNA polymorphism analysis is offered using Affymetrix SNP Chips, as well as direct DNA sequencing of selected SNPs after PCR amplification when required. Any reasonable quality DNA can be used for starting material. However, when the specimen contains less than 100 ug, pre-amplification using Qiagen DNA amplification services is indicated. The Core will assist users in procuring this service directly from Qiagen. The sample can then be sent to the user of the Core, at user discretion. Qiagen performs an initial QC analysis, with an estimate of “usability” for SNP analysis or DNA sequencing. If less than class I (highest quality), the user is contacted before further processing to ensure the user wishes to proceed. Sub-optimal call rates are then the responsibility of the user. If desired by the user, the sample is then processed along with all samples passing initial QC for analysis by SNP chips.
4. Gene Expression by QRT PCR : Frequently, users will wish to determine whether the expression values for a gene or group of genes are realistic estimates of actual expression values. The Core offers a confirmatory expression determination service, using quantitative real time PCR (QRT PCR). Up to 32 genes at a time can be processed using two Cepheid devices. Primer design by the Core is available. Samples from the hybridization mix (or fresh RNA) are run in parallel for beta actin and GAPDH, as well as for the gene of interest. The cycle threshold for control genes and unknown is determined, and relative quantification is determined by plotting Ct’s for known standards (beta actin and GAPDH) versus unknown. The expression level of the unknown can then be compared to the standards and compared to the results from microarray analysis.
5. SNP confirmation : Although ~95% of SNP calls are accurate when confirmed by other methods, there remains the problem of non-uniformity across a patient population or study set. Specifically, about 5% of the SNP calls on any given array may be ambiguous, and this 5% may vary across a study population (e.g., not the same 5% of SNPs). In order to confirm potential SNP associations with a co-variate of interest, such as disease susceptibility, phenotype, etc., it is sometimes useful to analyze the population for a specific SNP. In such cases, the Core will design suitable PCR primers, amplify the SNP in question from multiple individuals, and direct sequence the amplified sequence to identify the relative proportion of the target polymorphism. In addition, the Core is evaluating alternative methods based on solution hybridization for their potential utility. Finally, the Core is also actively evaluating methods better suited for regional interrogation of polymorphisms, in order to identify specific SNPs in a susceptibility locus.

      Policies and Procedures Manual (pdf)