Genotyping services are provided for investigators within and outside of the university through the CPPM Core Laboratory. Services are provided either as a price per sample or price per project depending on the specific needs of the investigator. Basic “a la carte” services, such as DNA isolation and quantitation are performed at a price per sample. Researchers interested in multiple services will be provided with an itemized estimated cost for the entire project. We strive to accommodate the needs of each researcher; if you do not see a service listed, please inquire.
All project estimates are projected totals; investigators will be notified of any price changes prior to work being completed. Price includes all reagents, consumables, and labor, unless otherwise noted. Data analysis is not automatically included in genotyping, gene expression, or DNA methylation projects. Analysis must be requested and is associated with additional cost.
Submitting Biological Samples
All samples must be delivered to the CPPM Core Lab in appropriate clearly labeled, sealed containers. Generally, samples are submitted in test tubes, vials or plates (96- or 384-well). All sample tubes or vials should be labeled, at minimum with a study ID and sample ID. All sample submissions should include an inventory file, preferably as a spreadsheet (using a program such as Microsoft Excel). The file should contain at least the following information for each sample:
- Study ID
- Sample ID
- Date of collection or aliquoting
- Location of sample: plate coordinates or box number
Importantly, to ensure that all work is done using appropriate informed consent processes, we require a copy of an IRB approval letter and a blank consent form under which the samples were collected. This document must be submitted before any work is undertaken.
DNA
The CPPM Core Lab can isolate DNA from buccal cells (cheek swab), blood, saliva, or tissue. If DNA has already been isolated and plated, please provide the DNA samples in clearly labeled 96-well plates. We reserve the last two wells of the 96-well plate (positions H11 and H12) for the controls so these should be left empty. If samples have not been quantified and normalized, we must do so using either the Quant-IT PicoGreen method of quantitation (see explanation of services) or standard UV absorbance. If DNA has already been quantitated, please normalize the DNA samples to ~20ng/ul if using the pyrosequencing or TaqMan methods. QuantStudio OpenArray genotyping requires a minimum of 50ng/ul. If submitting for send-out sequencing or a genome-wide array, we will communicate the desired concentration to assure submission requirements are met. Any remaining DNA will be returned after the genotyping is completed.
RNA
The CPPM can isolate RNA from blood or most tissue types. RNA is inherently less stable than DNA, requiring samples to remain frozen, with ideally no previous thawing. If samples have not been quantified and normalized, we must do so using either the Quant-IT PicoGreen method of quantitation (see explanation of services) or standard UV absorbance. Once normalized, the CPPM can convert RNA to cDNA for quantitative PCR (qPCR or sometimes called RT-PCR).
Transmission of Results
Data will be sent to the investigator electronically – generally in a spreadsheet format. Genotypes and default settings will be exported into an excel file as well as Hardy-Weinberg calculations. Gene expression measurements will be provided as cycle threshold (Ct) values. Additional raw data outputs can be provided upon request.
Platforms
We offer the following genotyping platforms at the UF Center for Pharmacogenomics. Our trained staff will help you select the platform that is best suited for your project.
Qiagen Pyromark Q48 Pyrosequencer™
Method of “sequencing in synthesis” that generates short sequence fragments up to 50 bases. Appropriate for SNP, INDEL, and microsatellite genotyping. The instrument can also be used for quantification of individual or multiple CpG sites in different genes (for DNA methylation analysis). Pyrosequencing method requires an initial assay design step that uses pyrosequencing software to design three compatible PCR and sequencing primers and to determine the quality of the assay. Standard and biotinylated oligonucleotides are then ordered for subsequent PCR and sequencing steps. For DNA methylation analysis, we can provide all steps in the process including bisulfite conversion of DNA samples.
QuantStudio™ 12K Flex System with OpenArray™, 384- and 96-well Capabilities
This method uses dye labeled probes to discriminate genotypes. This platform is also used for gene expression measurement (qPCR) and other real-time assays. Throughput is flexible – the system can accommodate OpenArray chips, TaqMan® array cards, 384-well, and 96-well plates. OpenArray chips can perform up to 3072 genotyping reactions simultaneously. These plates are customizable and ideal for researchers who have a large number of samples and SNPs to genotype.
Genotyping by the TaqMan method requires the selection of appropriate probes to assay the SNP of interest. Many SNP and gene expression assays are already commercially available and can be delivered within a week. Custom ordered assays may take significantly longer to obtain. The DNA requirements for OpenArray per sample is 20 ul of 50 ng/ul, all other applications require 20 ul of 20ng/ul.
Pharmacogenetic Analysis Services
The CPPM can also complete analyses on data derived from the CPPM Core Laboratory for an additional charge. This includes quality control analyses, data cleaning/processing, and statistical analysis. Statistical analyses include genetic association analyses and relative gene expression analyses. Investigators are encouraged to contact a biostatistician if advanced statistical analyses are required. Please inquire for specific methods and prices if CPPM data analysis is desired.