Services and Platforms

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.

flowchart.gifTotal 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. In order to recognize them, 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. 

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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).

(Image: Genomic20K - Image Genomic Image of the Core's 20k human Oligonucleotide microarray comparing MCF10 & UACC1179 breast cell line expression profile)

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 Microarrays

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

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 to discuss your experimental goals.

Custom Microarrays

The Core can manufacture custom microarrays in collaboration with your lab. Please contact us to 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.

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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 Bioarrays

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.

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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.

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.

Nimblegen Microarrays

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.

DNA: varies depending on application. Please contact the Core.

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.