Introductory information on microarrays can be found at
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.
The Core is continually updating and expanding its services
to keep up with advances in microarray technology and its applications.
Microarray analysis can be broken down into the following
components: array design and manufacture, experiment design, sample isolation,
target labeling, hybridization, data collection and archiving, and finally,
data confirmation. The Genomics Core provides solutions to all the steps
of microarray analysis including protocols for isolating samples, sample
quality control, Real-Time
PCR confirmation of results. The Core's microarray platforms,
applications, and associated services are broken down as follows:
Preparation:
Sample Quality Control
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 microarray platform. Research goals and budget
are also considerations in selecting samples, references, controls,
and sample isolation procedures. Questions regarding power and statistical
analysis should be referred to the Bioinformatics Shared Service (click
here to contact).
Applications for microarray:
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: spotted
cDNA microarrays, spotted oligonucleotide microarrays, Affymetrix
GeneChips, Agilent
microarrays, and Nimblegen
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.
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.
Services and Microarray Platforms
Spotted Microarrays: Oligonucleotide,
cDNA, CpG island, and Promoter Microarrays
Oligonucleotide
and cDNA spotted microarrays
By using the Core's cDNA-based
microarrays, the Principal Investigator consents to honor the 6 terms (a
through f) of the good faith agreement on use and distribution of IMAGE
consortium clones. A copy of this agreement can be found at: http://image.llnl.gov/image/html/GFA.html.
Total RNA or mRNA from two samples is isolated and
submitted by the researcher. The RNA is amplified by in vitro transcription
and labeled with Cy3 or Cy5. The fluorescently labeled samples are mixed,
and co-hybridized to the microarrays. Results are captured electronically
using a laser scanner that excites the fluorescent Cy dyes and quantitates
their emissions. For visualization purposes, Cy3 is assigned a green color
and Cy5 is assigned red. Equal amounts of red and green fluorescence emission
signal will result in a yellow color. Thus, deviation from yellow toward
red or green indicates differential expression of a particular gene between
the two samples. Raw data from hybridizations is archived and made available
in the researcher's web account. Analysis of the raw data can be performed
in one-on-one consultation with with George Watts. Contact
George Watts to set up a meeting to analyze microarray data.
The Core manufactures two types of spotted microarrays:
oligonucleotide-based and cDNA-based. The oligonucleotide-based
microarray is composed of 19,200 Sigma/Compugen 60-mers representing 18,600
human genes. The oligo library contains a probe for most well-defined genes
and several thousand expressed sequence tags (ESTs). The
cDNA-based microarrays are composed of 5,760 elements representing ~5,300
sequence-validated clones of human or mouse origin from the IMAGE
consortium. The microarrays are manufactured
by robotically spotting elements onto Corning UltraGAPS II slides (chemically
activated glass slides) using an OmniGrid printer produced by Gene Machines
and Stealth printing tips from Telechem International. The Core uses an
Axon Instruments GenePix 4000b laser scanner to capture and quantitate the
microarray hybridization results. The gene lists for the human and mouse
cDNA microarrays as well as oligonucleotide arrays are available for download
here. The
Core performs the RNA amplification, fluorescent labeling, hybridization,
scanning, quantitation, and analysis. Raw data is archived and posted to
the researcher's account on this website.
Sample Requirements:
Oligo-based and cDNA microarrays: 3 micrograms of
total RNA, or 0.3 micrograms mRNA in up to 8 microliters volume. cDNA
microarray (Human or mouse): 3 micrograms of total RNA, or 0.3 micrograms
mRNA in up to 8 microliters volume (i.e. 10 or 1 microgram(s)/microliter).
Notes for Sample Submission:
We require two RNA samples per hybridization, one
reference, and one “Test” in RNAse-free water.
We do not recommend lyophylization to concentrate RNA; use ethanol precipitation
instead. We recommend extracting RNA using the
Qiagen RNeasy kits, followed by ethanol precipitation to obtain the appropriate
concentration. Contaminants such as phenol will inhibit reverse transcription;
therefore we do not recommend the use of Trizol to extract your RNA unless
you are confident you can remove all phenol from your sample through chloroform
extraction and ethanol precipitation, or column purification.
For more information on submitting samples contact
us.
CpG Island (CGI) microarrays manufactured by the Core
are composed of ~7,000 cloned CpG-rich genomic fragments. The sequenced
clones are PCR amplified and printed as microarrays in the same manner as
cDNA-based arrays. The CGI arrays can be used in ChIP
on chip experiments to query DNA methylation, chromatin structure, and
transcription factor binding. The CGI microarrays are offered as a full
service for ChIP
on chip analysis of CpG methylation only. For other applications the
Core supports researchers from the hybridization step onwards i.e. the researcher
performs sample isolation by chromatin immunoprecipitation and labeling.
The CGI microarrays are available for purchase in sets of 10 arrays for
researchers wishing to perform hybridizations themselves. The Core supports
such researchers with access to scanners, detailed protocols, expertise,
data storage, and analysis software. Please contact
us discuss your experimental goals.
Promoter microarrays manufactured by the Core are
composed of ~10,000 promoter sequences made by PCR-amplification. The promoter
sequences are typically 500-1500 bases in length and are associated with
the 5' ends of well annotated genes. The promoter microarrays can be used
in ChIP
on chip experiments to query chromatin structure, transcription factor
binding, DNA methylation, etc. The Core supports the researcher from the
hybridization step onwards i.e. the researcher performs sample isolation
by chromatin immunoprecipitation and labeling. The CGI microarrays are available
for purchase in sets of 10 arrays for researchers wishing to perform hybridizations
themselves. The Core supports such researchers with access to scanners,
detailed protocols, expertise, data storage, and analysis software. Please
contact
us discuss your experimental goals.
The Core can manufacture custom microarrays in collaboration
with your lab. Please contact
us discuss your needs.
Commercial 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
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, 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 Biooarrays
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.
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.
The Core has implemented the Nimblegen microarray platform
as of August 2007. Nimblegen microarrays combine advantages of having multiple
probes per target like Affymetrix GeneChips with the flexibility to produce
custom microarrays at no extra charge like the Agilent microarrays. The
Nimblegen microarrays consist of short oligos like those found on the Affymetrix
platform, but printed on modified glass slides rather than a silicon wafer.
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.
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.
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, the Southwest Environmental Health Science Center, and the BIO5
institute, we must recover costs not covered by these supporting institutions
as follows (costs are per sample or array):
RNA expression
cDNA Microarray Expression Analysis:
human or mouse "5k" cDNA microarray (~5,300
genes)
$99.00
Full service hybridization
$150.00
Oligonucleotide Microarray Expression
Analysis:
human "20k" oligonucleotide microarray (~18,600 genes)
$99.00
Full service hybridization
$150.00
Affymetrix Expression Analysis:
Affymetrix GeneChip, 3' or ST (Chip only)
Variable (range is $175-400)*
Full service hybridization - 3' expression GeneChips (reagents only)
$325.00
Full service hybridization - ST expression GeneChips (reagents only)
$325.00
Full service hybridization ST expression GeneChips plus
reagents - 10-pack
$425.00
Agilent Expression Analysis:
Agilent microarray
Variable (range is $100-400)*
Full service hybridization
$325.00
Nimblegen Expression Analysis:
Nimblegen microarray
Variable (range is $100-400)*
Full Service hybridization
$325.00
RNA Regulation
CpG Island Analysis:
CpG Island microarray
$99.00
Full service hybridization, methylated CpG ChIP on chip only
$200.00
Bundle of 10 CGI microarrays
$750.00
Promoter Microarrays:
hybridization and data acquisition
$80.00
Bundle of 10 promoter microarrays
$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, Agilent, Nimblegen microarrays for
CGH, re-sequencing, and SNP analysis
Variable*
CGH hybridization and data acquisition
$80.00
re-sequencing hybridization and data acquisition
$80.00
SNP analysis hybridization and data acquisition
$80.00
Self-service Scanning
Self service laser scanning of user-hybridized slides
$20.00 per 30 minutes scanner use
Custom microarray slide fabrication
Variable**
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
RNA integrity and quantitation
$8.00 per sample
DNA integrity and quantitation
$8.00 per sample
*Affymetrix, Agilent, and Nimblegen microarrays
must be purchased from the manufacturer and provided by the user. Please
contact
the Core for information on ordering and prices for specific microarrays.
**Prices for custom microarray manufacturing depend on
the size and type of microarray, please contact
the Core for a quote.
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 analysis of my sample does not work?
A: 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 gene lists for the human and mouse cDNA
and oligonucleotide arrays are available here.
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 GSS also archives the raw image
files from each hybridization should you need access to them later.
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 from the Core 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.
11) 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.
12) Q: Will you help me analyze my data?
A: Yes. We recognize that microarray data analysis
poses a challenge for our users. Interested users are encouraged to
contact George Watts
for consultation and analysis using Silicon Genetics' GeneSpring software.
13) 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
14) 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 For more
information, please contact Amber May (520.626.0267).
15) 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
you can calculate relative expression levels between your samples.
