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Genomics Core
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Prices
DNA Sequencing (using either the Ion Torrent PGM or Proton)
Next-Gen Sequencing:
Human Whole Exome Sequencing* $950
46 gene cancer panel mutation analysis $600.00
Custom gene panel sequencing $300-600
RNA Expression
Affymetrix Gene Expression Analysis:
Affymetrix GeneChips$175-400**
Affymetrix hybridization$250-325
RNA Regulation
Epigenetic Analysis:
ChIP on chip DNA methylation analysis (chip plus hyb)$750
ChIP on chip histone acetylation analysis (chip plus hyb)$750
miRNA expression analysis$400
DNA analysis
Affymetrix and Agilent chips for CGH, re-sequencing, and SNP analysis$150-300
Agilent aCGH$325
Affy genotyping$325
Self-service Microarray Scanning
Self service laser scanning of user-hybridized slides$20.00 per 30 minutes
Real-Time PCR or RT/PCRSelf-service RT/PCR or PCR $50 per plate
full service RT/PCR or PCR $12 per reaction, includes RT
Sample Quality Control Agilent Bioanalyzer
$16 full service
Nanodrop spectrophotometry$12/$4 full service/self service
*The Genomics Core is a member of the "certified service provider" program.

**To avoid maintaining 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.


How to Submit A Sample.
Obtain a good quality sample:It is critical to provide high quality RNA or DNA as the starting material for genomic analyses. Common problems with samples include: RNA or DNA degradation, incorrect concentration, contaminants (proteins, salt, phenol, etc), and finally the presence of RNA in DNA preparations or vice versa. To help you provide good quality samples, we recommend isolation kits available from Qiagen. For total RNA isolation use the RNeasy line of kits specific to your sample type. RNA yields vary, but 1 million cells in culture will yield approximately 10-30 micrograms of total RNA. Ten milligrams of tissue will yield approximately 20-80 micrograms of total RNA. For genomic DNA isolation use the QIAamp kits. DNA yields vary, but are approximately 3 micrograms per 1 million cells in culture. Ten milligrams of tissue will yield approximately 5-20 micrograms of genomic DNA. Qiagen kits are available to University of Arizona researchers from University Stores.

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.

Finally, we have written a guide to interpretation of spectrophotometric results that is available here.

Submit sample: First, contact us to discuss your research goals, budget, etc - we're here to help! Create a user account/profile on this website and order any microarrays that will be needed (we help with the choosing and ordering of arrays). Log in to your account and select the "Submit Sample Request" link and fill in the requested information. You will receive an email confirming your request. Bring your samples and your microarrays (if applicable) to the lab. Call the lab at (520) 626-0267 to make sure someone will be available to take your samples, and then bring them to the lab.


Notification of Results and Data Analysis.

A typical experiment will result in raw data files ready for bioinformatic analysis, for example DNA sequencing will result in .bam files while an Affy microarray analysis will result in .cel files. Data files are uploaded by the Core to user's accounts on this website and users are notified by e-mail when their data is available for download. The Core recognizes that many users will want or need help performing data analysis. For simple experiments, the core will either provide analysis (e.g. full service PCR, RT-PCR) or point the user to software allowing the user to perform analysis themselves (e.g. Affymetrix Transcriptome Analysis Console or BRB ArrayTools). For more complex analysis (e.g. pathway analysis) users can contact the Bioinformatics Shared Service


Frequently Asked Questions

  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.


  4. Q: There's a lot of different Affy expression arrays out there: Gene ST, Exome ST, HTA 2.0, what's the difference and which should I pick?
    A: Here's an overview. The choice of which array or platform to choose is important and we can help you choose, contact us for assistance.


  5. Q: How is my data returned to me?
    A: Your raw data will be archived on the password protected portion of this web site.


  6. Q: Will you help me analyze my data?
    A: It depends, for real time PCR results, we will do it for you. You can do the analysis yourself with guidance from us using free-ware such as Affymetrix Transcriptome Analysis Console or BRB ArrayTools). For more complex analysis (e.g. pathway analysis) users can contact the Bioinformatics Shared Service


  7. Q: Can I buy microarrays and reagents and do the hybridizations myself in your lab?
    A: Yes, but genomic analyses can have an expensive learning curve and therefore do not reward the casual practitioner. If you believe you will be doing a significant amount of microarray work, we can arrange to access to the lab, training, and protocols.


  8. Q: What do you charge for arrays, hybs, sequencing, real time PCR, etc.?
    A: Our pricing is listed above.


  9. 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


  10. 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.


What are Microarrays? Introductory information on microarrays can be found here.

What is sequencing? Introductory information on sequencing can be found here.


About


Services

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, and confirmation of results. The Core's platforms, applications, and associated services are broken down as follows:
  • Sample Quality Control: If you are performing your own quality control using spectrophotometry, 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 the appropriate personnel to answer questions regarding power and statistical analysis.


  • Applications:

    • Sequence analysis: The core has two Ion Torrent Personal Genome Machines and an Ion Torrent Proton, offering next-gen sequencing capabilities for large and small sequencing projects. Currently, we offer both 200 and 400 base pair read lengths utilizing PGM 314, 316, and 318 chips and 200 base pair sequencing with the P1 chips for the Proton. P2 chips capable of whole human genome sequencing are expected to be released this year (2014). DNA Sequencing can be used to look for mutations in FFPE tumor samples (e.g. 46 gene Ampliseq cancer panel), examine whole exome sequence variation (did you know we're a certified exome service provider?), measure gene expression (e.g. RNAseq), identify bacteria in a sample (e.g. 16s rRNA sequencing), even analyze copy number variation.


    • 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 GeneChipsand Agilent 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.


    • 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: Affymetrix GeneChips and Agilent microarrays. As with RNA expression analysis, the Core will consult with the user to match experimental goals with the appropriate platform.


Equipment Platforms
  • Ion Torrent Next-Gen Sequencing: The Core is a member of the Ion Torrent Certified Exome Service Provider Program which ensures users high quality human exome sequencing with short turn around time. The Core has two types of Ion Torrent sequencers, both are based on a unique approach to sequencing that depends on detecting the release of protons during the incorporation of a nucleotide during template-directed DNA polymerization.Image of an Ion Torrent Proton

    Ion Torrent's approach requires no modified nucleotides and does not require laser excitation or fluorescence detection by CCD cameras to work. This allows the equipment to be simpler, resulting in fast and cost-effective sequencing. Currently the Core has two Ion Torrent Personal Genome Machines as well as an Ion Torrent Proton. Between the two machines, the Core has the capacity to sequence as little as 50 Megabases out to 13 Gigabases in a single run, with either 200 or 400 base pair reads. DNA libraries can be prepared for sequencing from many sources including genomic DNA (e.g. whole genome sequencing), selected DNA fractions (e.g. Chromatin Immunoprecipitated DNA, mitochondrial DNA, exomic DNA), RNA (e.g. RNAseq), and PCR amplicons (e.g. Ampliseq). Sample requirements vary by application, but are typically less than 100 nanograms. The Core has had success sequencing DNA derived from samples preserved by FFPE (formalin-fixed, paraffin-embedded) using the amplicon-based apporach. Before starting any sequencing project for the Ion Torrent system it is highly recommended that users contact the Core to discuss their goals.

    Sample Requirements: Exome sequencing: 100 nanograms in a maximum volume of ten microliters. For other applications, please contact the Core to discuss your project.



  • Affymetrix GeneChips: Affymetrix GeneChips are commercially manufactured microarrays made by directly synthesizing oligonucleotides on a silicon chip via photolithography. Image of an Affymetrix GeneChip

    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 steps of the microarray process after RNA isolation. For DNA-based applications (e.g. ChIP on chip, CGH, re-sequencing, SNP analysis), the Core perform all steps after DNA isolation. 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: 100 nanograms in a maximum volume of ten microliters. DNA: varies depending on application, contact the Core to discuss your application.



  • 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.Agilent microarrayThe 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 ten microliters. mRNA: 0.1 to 5 micrograms in a maximum volume of ten microliters. DNA: for aCGH, we need 100 nanograms in 10 microliters.



  • 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. Alternatively, 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 fluorescently 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 50 nanograms in a maximum volume of ten microliters.
    The minimum amount of starting mRNA is 5 nanograms in a maximum volume of ten microliters. The minimum amount of starting genomic DNA is 50 nanograms in a maximum volume of ten microliters.