What Are Microarrays?
Introductory information on microarrays can be found here.
What is sequencing?
Introductory information on sequencing can be found here.
Our Name(s)
The Genomics Shared Service/Genomics Facility Core is referred to by different names in the grants that support it, but will be referred to as the Genomics Core, or just "the Core", on this web page.
Overview Of the Service
The Core is continually updating and expanding its services to keep up with advances in microarray and sequencing technology and its applications. We try to complete service requests within two weeks, dependent on back logged requests and user priority. The Genomics Core provides solutions to all the steps of microarray analysis and sequencing, including protocols for isolating samples, sample quality control, Real-Time PCR confirmation of results. The Core's platforms, applications, and associated services are broken
down as follows:
Preparation:
Sample Quality Control
If you are performing your own quality control using spectrophotmetry, we have written a guide to interpretation
here. The guide is specific to the NanoDrop spectrophotometer but nearly all of the information is applicable to spectrophotometry on any instrument. RNA and DNA samples are checked for quality and quantity. The Core checks every sample before use to ensure success of the microarray analysis. Samples are run on the Agilent Bioanalyzer which simultaneously quantitates the sample as well as measuring sample integrity. In the case of RNA, sample integrity is quantitated as a
RNA integrity number (RIN).
Experiment Design
The Core provides free consultation aimed at defining experimental goals and then matching those goals to the researcher's budget and appropriate technology. Research goals and budget are also considerations in selecting samples, references, controls, and sample isolation procedures. We can also refer you to teh appropriate personnel to answer questions regarding power and statistical analysis.
Microarray Applications:
RNA expression
The original application of microarrays, expression analysis is the application for which the researcher has the most options. The Core offers "complete service" for analysis of RNA expression meaning that the researcher need only provide sample RNA and the Core handles all other aspects of the process: sample quality control, labeling, purification, hybridization, data collection, data analysis, and data archiving. Researchers have the following platforms from which to choose: Affymetrix GeneChips and Agilent microarrays. These platforms have been chosen to serve the widest possible research community. A key part of the service is consultation with the researcher to determine which platform is appropriate for their goals during the preparation stage (see above).
RNA regulation
By designing probes to non-coding regions of the genome, microarrays can be used to survey genomic DNA with chromatin immunoprecipitation
ChIP on chip) and comparative genomic hybridization (
CGH). ChIP on chip and CGH can detect factors affecting RNA expression including: transcription factor binding, chromatin structure, DNA methylation, copy number, deletions, and amplifications. The Core provides full service for researchers wishing to use the Core's CpG island microarrays for DNA methylation analysis. For all other microarrays the researcher will be responsible for isolating the genomic DNA sample and labeling it for hybridization. The Core will then perform the remainder of the process from the hybridization onwards. Researchers have the following platforms from which to choose: spotted cDNA microarrays, spotted oligonucleotide microarrays, Affymetrix GeneChips, Agilent microarrays, and Nimblegen microarrays. As with RNA expression analysis, the Core will consult with the user to match experimental goals with the appropriate platform.
DNA analysis
Several platforms offered by the Core can be used by the researcher to compare DNA sequences between samples to identify sequence variation (re-sequencing arrays), including single nucleotide polymorphisms. Platforms to choose from include Affymetrix GeneChips, Agilent microarrays, and Nimblegen microarrays. As with applications for analysis of RNA regulation, the researcher will isolate the genomic DNA sample and prepare it for hybridization at which point the Core will perform the remaining steps.
Sequence analysis
The Ion Torrent Personal Genome Machine can be used to analyze sequence variation in your samples. The platform offers a Cancer Panel that identifies mutations in 46 cancer related genes. In addition, custom panels can be created to look at sequence variation in any portion of any known genome.
Data analysis and confirmation:
Data analysis
Results are loaded to the Core's website and user's are notified automatically that their data are ready. Analysis of microarray data is offered by the Bioinformatics Shared Service at the Arizona Cancer Center. (click here to contact)
Data confirmation
The Core provides a full real time PCR service for confirmation of microarray results. Real time RT/PCR to confirm differences in RNA expression can be performed using the ABI TaqMan Gene Expression Assays or a locked nucleic acid Universal Probe Library from Roche. Real time PCR can also be used to confirm results found by DNA based applications such as ChIP on chip and CGH.
Recommendations for Sample Preparation.
It is critical to provide high quality RNA or DNA as the starting material for microarray analysis. Problems we have found with user samples include: RNA or DNA degradation, incorrect concentration,and contaminants (proteins, salt, phenol, etc), and finally the presence of RNA in DNA preparations or vice versa. To help you provide good quality sample, we recommend isolation kits available from Qiagen. For total RNA isolation use the RNeasy Mini or Midi kit. RNA yields vary, but 10 million cells in culture will yield approximately 100-300 micrograms of total RNA. Ten milligrams of tissue will yield approximately 20-80 micrograms of total RNA. For genomic DNA isolation use the QIAamp DNA Mini or Midi kit. DNA yields vary, but are approximately 30 micrograms per 10 million cells in culture. Ten milligrams of tissue will yield approximately 5-20 micrograms of genomic DNA.These Qiagen kits are available to all University of Arizona researchers from University Stores. Finally, we have written a guide to interpretation of spectrophotometric results that is available here.
Services and Microarray Platforms
Affymetrix GeneChips.Affymetrix GeneChips are commercially manufactured microarrays made by directly synthesizing oligonucleotides on a silicon chip via photolithography.
Each target sequence to be queried is represented on the GeneChip by multiple probes to increase the robustness of the data. Affymetrix offers GeneChips for a large and expanding variety of applications and model organisms. Affymetrix GeneChips are "single-color" arrays - meaning that only one sample is hybridized per chip as opposed "two-color" competitive hybridizations (e.g. Cy3 versus Cy5). Depending on the application, researchers supply sample RNA or DNA along with the GeneChip for analysis. For RNA expression, the Core performs all subsequent steps of the microarray process including data analysis and data archiving. For DNA-based applications (e.g. ChIP on chip, CGH, re-sequencing, SNP analysis), the Core will perform all steps after sample preparation. The Core will assist the researcher in performing analysis of the results which can be posted on this web site or emailed to the investigator. Before starting any work with the Affymetrix GeneChip system it is highly recommended that users visit the Affymetrix website and contact the Core to discuss their goals.
Sample Requirements:
Total RNA: One to ten micrograms depending on the GeneChip in a maximum volume of nine microliters. mRNA: 0.1 to 1 micrograms in a maximum volume of nine microliters. DNA: varies depending on application. Please contact the Core or consult the relevant application manual on the Affymetrix websiteNotes for Sample Submission: Samples should be dissolved in RNAse-free water. We do not recommend lyophylization to concentrate RNA, use ethanol precipitation instead. Contaminants such as phenol will inhibit reverse transcription reactions, consequently, we request that you do not use trizol to extract your samples unless you are confident you can remove all contaminants through subsequent chloroform extraction and ethanol precipitation or column purification. For more information on submitting samples contact us.
Agilent Arrays.
Agilent microarrays are commercially manufactured oligonucleotide-based microarrays made by using ink jets to synthesize long oligonucleotides on modified glass slides. Because of flexibility in the manufacturing technology Agilent Technologies will produce custom microarrays to query any set of user defined target sequences for the same price as one of its catalog arrays with no minimum order size. Agilent microarrays can be used in "single-color" and "two-color" hybridizations and are offered for a variety of applications and model organisms. Agilent microarrays can also be multi-plexed, that is, more than one microarray is synthesized on a single glass slide and gaskets are used to separate the microarrays into separate chambers. By allowing two, four, or more separate hybridizations to be performed on a single slide, user costs are reduced.
The researcher supplies sample RNA or DNA for the microarray analysis. For RNA expression, the Core performs all subsequent steps of the microarray process including data analysis and data archiving. For DNA-based applications (e.g. ChIP on chip or CGH), the Core will perform all steps after sample preparation. The Core will assist the researcher in performing analysis of the results. Before starting any work with Agilent microarrays it is highly recommended that users visit the Agilent Technologies website for more information about the microarrays offered and contact the Core to discuss their goals.
Sample Requirements:
Total RNA: 1 to 5 micrograms in a maximum volume of nine microliters. mRNA: 0.1 to 5 micrograms in a maximum volume of nine microliters. DNA: varies depending on application. Please contact the Core or consult the relevant application manual on the Agilent Technologies website
Notes for Sample Submission: Samples should be dissolved in RNAse-free water. We do not recommend lyophylization to concentrate RNA, use ethanol precipitation instead. Contaminants such as phenol will inhibit reverse transcription reactions; therefore we request that you do not use Trizol to extract your samples unless you are confident you can remove all contaminants through chloroform extraction and ethanol precipitation, or column purification. For more information on submitting samples contact us.
Real Time PCR and RT-PCR
The Real Time PCR and RT/PCR service is offered using the Applied Biosystems (ABI) 7000 and 7300 SDS machines. For real time RT/PCR applications, ABI has designed primer/probe sets for most exons of human, mouse, and rat and offers them through the Gene Expression Assays program. The Genomics Shared Service will reverse transcribe and amplify the sample RNA and analyze the results using primer/probe sets purchased by the investigator from ABI. For real time PCR and RT/PCR applications the researcher may also choose to use probes from Roche's Universal Probe Library to reduce costs. In addition, researchers are welcome to use the real time PCR machines in a self-service fashion for a flat fee per plate.
Real-time PCR depends on a fluoresecently tagged probe designed to bind the PCR template between the PCR primers. Attached to the 5’ end of the probe is a fluorescent reporter dye; on the 3’ end of the probe is a non-fluorescent quencher. When the reporter and the quencher are in close proximity to each other, the fluorescence from the reporter is quenched. When the Taq polymerase encounters the probe during extension, it digests the probe and releases the reporter from its close proximity to the quencher. After each round of PCR fluorescence is detected in each reaction tube. The result is a logarithmic amplification plot that shows the intensity of fluorescence over the number of cycles in the PCR reaction. A threshold intensity value is set in the log-linear portion of the amplification curve and each sample is given a cycle threshold (Ct) value. The Ct value corresponds to the cycle at which the amplicon reached the selected threshold of fluorescent intensity, which is equivalent to a certain amount of PCR product. The lower the Ct value the more copies of cDNA were present as template, which corresponds to the amount of a gene’s RNA that was present in the sample. Because the real-time PCR reaction run by the Core is comparative, a reference gene is used to correct for experimental error. Common reference genes used to measure baseline expression are GAPDH, Beta-actin, and Beta-glucuronidase, all of which are available from the Core service.
Sample Requirements:The minimum amount of starting total RNA is 200 nanograms in a maximum volume of ten microliters.
The minimum amount of starting mRNA is 20 nanograms in a maximum volume of ten microliters. The minimum amount of starting genomic DNA is 200 nanograms in a maximum volume of ten microliters.
Notes on Sample Submission:
Samples should be dissolved in RNAse-free water. We do not recommend lyophylization to concentrate RNA, use ethanol precipitation instead. Contaminants such as phenol will inhibit reverse transcription reactions; therefore, we request that you do not use Trizol to extract your samples unless you are confident you can remove all contaminants through chloroform extraction and ethanol precipitation, or column purification.For more information on submitting samples contact us.
Although we receive support from the Arizona Cancer Center and the Southwest Environmental Health Science Center we must recover costs not covered by these supporting institutions as follows (costs are per sample or array):
Ion Torrent Sequencing
Cancer Panel Mutation Analysis:
314, 316, 318 sequencing chip
$99, $299, $499
46 gene cancer panel mutation analysis
$600.00 per sample when multiplexing 4 samples/run
Custom sequencing projects
Variable
RNA expression
Affymetrix Expression Analysis:
Affymetrix 1.0 ST GenChips
Variable (range is $175-400)*
Affymetrix GeneChip hybridization
ST GeneChips: $200; all others: $325
RNA Regulation
DNA methylation analysis:
Full service hybridization, methylated CpG ChIP on chip
$750.00
Affymetrix, Agilent, and Nimblegen microarray for ChIP on chip (transcription factor binding, chromatin structure, etc)
Variable*
hybridization and data acquisition
$80.00
DNA analysis
Affymetrix and Agilent chips for
CGH, re-sequencing, and SNP analysis
Variable*
re-sequencing hybridization and data acquisition
$80.00
SNP analysis hybridization and data acquisition
$80.00
Self-service Microarray Scanning
Self service laser scanning of user-hybridized slides
$20.00 per 30 minutes scanner use
Real-Time PCR or RT/PCR
self-service RT/PCR or PCR
$35.00 per plate
full service RT/PCR or PCR
$8.00 per reaction, includes RT
Sample Quality Control
Agilent Bionalayzer
$8.00 full service
Nanodrop spectrophotometry
$8.00/$4.00 full service/self service
*To avoid maintianing an inventory of chips that expire, the Core will assist users in purchasing their Affymetrix and Agilent microarray chips from the manufacturer. Please contact the Core for information on ordering and prices for specific microarrays.
Contact Information
Laboratory Address:
Genomics Shared Service, Room 3933 Arizona Cancer Center 1515 N. Campbell Ave. Tucson AZ 85724
A typical hybridization will result in multiple raw data files. Users are notified by e-mail when the raw data is available for download from their account on this web site. The Core recognizes that many users will want help performing data analysis. Data analysis is provided through the Bioinformatics Shared Service Researchers wishing to meet individually for data analysis and consultation should contact David Mount to schedule an appointment once all the relevant hybridizations have been completed. The Bioinformatics Shared Service website is here.
1) Q: I can't get a lot of RNA from my samples. What can I do?
A: Two rounds of in vitro transcription can be used to amplify very small RNA samples. The Core does not currently offer this as a service, but prior users have been successful performing this procedure themselves and then submitting the labeled sample for hybridization to us.
2) Q: I want to analyze specific cell types from a tissue specimen, is this possible?
A: Yes, you will need use Laser Capture Microdissection(LCM) first, then isolate and amplify the RNA. Contact the Tissue Acquisition Shared Service for access to LCM instrument and contact the Core to discuss you experiment.
3) Q: Will you charge me if the microarray hybridization/Real Time PCR/Sequencing analysis of my sample does not work?
A: Typically, no. Our goal is to provide quality data to further your research goals. We will only charge you for hybridizations that work. The most likely source of failure is poor quality sample - this is why the Core runs all samples through quality control before using them. The second most likely source of failure is manufacturing defects in the microarrays or the kits we use to run them. The manufacturers usually reimburse the researchers for these types of failures.
4) Q: Does the Genomics Shared Service perform RNA amplifications on investigator provided samples?
A: Yes, single round amplification is integrated into all our expression platforms.
5) Q: Where can I get a list of the genes on the Core's spotted cDNA and oligonucleotide microarrays?
A: The Core no longer manufactures microarrays. Genelists for the commercial arrays we offer (Affymetrix and Nimblegen) are available on teh munfacturer's websites.
6) Q: How is my data returned to me?
A: Your raw data will be archived on the password protected portion of this web site. The Core also archives Data analysis can be performed in consultation with the Bioinformatics Shared Service (click here to contact).
7) Q: Will you help me analyze my data?
A: Yes - the Bioinformatics Shared Service will. We recognize that microarray data analysis poses a challenge for our users. Interested users are encouraged to contact David Mount to set up an appointment to analyze their data.
8) Q: Can I buy microarrays and do the hybridizations myself?
A: Yes. Protocols for fluorescent target preparation are available. Please contact George Watts. We have found that the microarray process can take time to learn and get working well. Thus, the technique does not reward the casual user. If you believe you will be doing a significant amount of microarray work, we can arrange to provide arrays, access to the scanner, training, and protocols.
9) Q: How much do Affymetrix/Agilent/Nimblegen arrays cost?
A: If you are a user at an academic institution in the state of Arizona we can provide you with the academic pricing for the microarrays offered by our commercial platforms. See our price list above.
10) Q: Where do I get the primer/probe sets for real time RT/PCR for a gene of interest?
A: Assays can be searched for in the Assays-on-Demand section of the ABI web site
11) Q: My gene(s) of interest are not available from Assays-by-Design, now what?
A: If your gene of interest does not have an assay-on-demand yet, you can request an assay (primer pair) be made for you via the Assays-by-Design portion of the Applied Biosystems web site.
12) Q: What kind of results do I get back from the real time RT-PCR service?
A: Once your samples have been run you will receive an amplification plot of your data and a report showing the cycle threshold (Ct) of each sample and your controls. Using this information we can calculate relative expression levels between your samples.
The researcher supplies sample RNA or DNA for the microarray analysis. For RNA expression, the Core performs all subsequent steps of the microarray process including data analysis and data archiving. For DNA-based applications (e.g. ChIP on chip or CGH), the Core will perform all steps after sample preparation. The Core will assist the researcher in performing analysis of the results. Before starting any work with Agilent microarrays it is highly recommended that users visit the 
George Watts, Ph.D, Co-Director
Bernard Futscher, Ph.D, Co-Director
Candice Clark, Research Technician
Gregory Metzger, Research Technician