University of Pittsburgh Cancer Institute (UPCI)

CCSG Acknowledgement

Required CCSG Acknowledgement

The NCI requires that publications acknowledge the UPCI CCSG support, and they are tracking compliance. If a UPCI CCSG-supported Shared Resource provided data used in your publication, please include the following statement in the acknowledgment section of your publication(s):

"This project used the UPCI [insert name(s) of shared resource(s)] that [is/are] supported in part by award P30CA047904."

Shared Resource Directors: Please make sure to include this statement on all of your order forms, contracts, etc. as a reminder to your users to acknowledge the UPCI CCSG support.


RNA Expression: Affymetrix

Click here to order an Affymetrix GeneChip expression assay.

Agilent Bioanalizer

The CBF utilizes several different commercial assays to generate hybridization targets for Affymetrix microarray studies, depending on the type and quality of the input samples.

The Ambion MessageAmp Premier and the Ambion WT Expression Labeling assays are used to generate hybridization targets from high quality, highly abundant RNA samples.

The NuGen Ovation FFPE WTA assay is used to generate hybridization targets from degraded RNA samples. Investigators may choose to have the CBF extract and prepare RNA for them directly from frozen tissues or cell samples, FFPET samples, or they can choose to submit investigator-prepared RNA.

High quality RNA is critical to the success of any microarray expression assay, so all samples generated within the CBF and investigator-prepared samples will be assessed for quality via the Nanodrop spectrophotometer and the Agilent Bioanalyzer. Investigators will be notified of all samples that do not meet Quality Control criteria for quantity and quality. Please see Sample Requirements below for more information.

Affymetrix whole genome expression arrays are available for human, mouse, rat, and many other eukaryotic and prokaryotic model organisms. The Affymetrix whole transcript expression arrays, which assay alternatively spliced transcripts, are available for human, mouse, and rat.

Human Expression Arrays

3' arrays (IVT antisense chemistry - Ambion MessageAmp Premier assay)

  • 133A 2.0
    • 18,400 transcripts and variants, including 14,500 well-characterized human genes
    • Comprised of more than 22,000 probe sets and 500,000 distinct oligonucleotide features
  • U133 Plus 2.0
    • Complete coverage of the U133A 2.0 transcripts plus 6,500 additional genes for analysis of over 47,000 transcripts

Whole transcript arrays (sense strand chemistry - Ambion WT Expression assay)

  • Gene 1.0 ST
    • interrogates 28,869 well-annotated genes with 764,885 distinct probes
    • uses a subset of probes from the Exon 1.0 ST Array and covers only well-annotated content
    • design based on the March 2006 (UCSC hg18, NCBI Build 36)
  • Exon 1.0 ST
    • Highest resolution expression analysis, interrogating over 1 million exon clusters within the known and predicted transcribed regions of the entire genome
    • Probe Sets - 1.4 million
    • Exon Clusters - >1 million

Mouse Expression Arrays

3' arrays (IVT antisense chemistry - Ambion MessageAmp Premier assay)

  • 430A 2.0 - 14,000 well-characterized mouse genes
  • 430 2.0 - over 39,000 transcripts

Whole transcript arrays (sense strand chemistry - Ambion WT Expression assay)

  • Gene 1.0 ST - 28,853 genes
  • Exon 1.0 ST
    • Probe Sets - 1.2 million
    • Exon Clusters - ~ 1 million

Rat Expression Arrays

3' arrays (IVT antisense chemistry - Ambion MessageAmp Premier assay)

  • 230 2.0 - Comprised of more than 31,000 probe sets, analyzing over 30,000 transcripts and variants from over 28,000 well-substantiated rat genes
  • Whole transcript arrays (sense strand chemistry - Ambion WT Expression assay)
    • Gene 1.0 ST - more than 27,000 gene-level probe sets
    • Exon 1.0 ST
      • Probe Sets - 1 million
      • Exon Clusters - 850,000

Sample Requirements


Sample Type Expression Array Type Target Prep Method *^Minimum Amount of Total RNA Required Minimum Concentration
Fresh 3' IVT Ambion MessageAmp Premier Kit 500ng 166.67ng/ul
Fresh 3' IVT NuGEN 50ng-100ng  
Fresh Gene ST 1.0 Ambion WT Expression Kit 500ng 166.67ng/ul
Fresh Gene ST 1.0 NuGen 50ng-100ng  
Fresh Exon 1.0 ST Ambion WT Expression Kit 500ng 166.67ng/ul
Fresh Exon 1.0 ST NuGen 50ng-100ng  
FFPET 3' IVT NuGen 100ng 33.34ng/ul
FFPET Gene ST 1.0 NuGen 100ng 33.34ng/ul
FFPET Exon 1.0 ST NuGen 100ng 33.34ng/ul
*Quality, Quantity, and Concentration subject to verification by the CBF
^ These are the minimal amounts specified in the respective protocols. We recommend you provide us with excess in order to run QC analysis and to repeat assays if necessary

High quality array data depends upon high quality samples!

RNA: Prior to target synthesis, the CBF will evaluate all submitted RNA samples for quantity and quality (amount of degradation). RNA will be quantified by UV absorbance using a Nanodrop spectrophotometer and validated to be non-degraded via the Agilent Bioanalyzer. A260:A280 ratios should be 1.8-2.0 and the RIN should be >7.0.Submit RNA in nuclease-free water.

Tissue or cells may also be submitted to the CBF for extraction prior to performing an expression array assay. For researchers who are collecting tissue on their own, we suggest a few guidelines to limit RNA degradation and ensure sample quality. All samples submitted to the lab for extraction and downstream microarray applications should be handled in one of the following ways at the time of collection:

  • Fresh tissue should be flash frozen in 2.0 mL or Nunc tubes in LN2 or dry-ice and maintained at -80°C until submitted to the CBF. Cells should be pelleted in 1.5-2.0 mL tubes, all supernatant removed, and then flash frozen in LN2 and maintained at -80°C until submitted to the CBF.
  • Tissue and cell pellets should be placed in RNAlater (Ambion, Inc) and stored at 4°C for up to one month, or
    Tissue and cell pellets should be placed in RNAlater at 4°C overnight, then stored at -20°C indefinitely until submitted to the CBF, or
    Tissue should be placed in RNAlater at 4°C overnight, then removed from the RNAlater and stored at -80°C indefinitely until submitted to the CGIF. Cell pellets should be placed in RNAlater at 4°C overnight, then stored at -80°C indefinitely until submitted to the CBF.
  • Tissue and cell pellets should be homogenized immediately in either Qiagen Buffer RLT (recommended) or Trizol (not recommended) and then stored at -80°C until submitted to the CBF.

Once samples have been extracted they will be assessed for quality, quantity, and concentration prior to beginning microarray assays.

Investigators will be notified if a sample is of insufficient quantity or quality (failed Quality Control parameters) for optimal array performance.

Samples cannot be processed until a completed Service Request Form is received along with a signed estimate. Click here for pricing information.

If you would like to order an array that is not listed, please contact the CBF.

Technology and Equipment

Affymetrix 3' IVT Arrays

Affymetrix 3' IVT Arrays

View a complete list of Affymetrix 3' IVT Arrays that can be run in the CBF.

Most commonly used arrays in the CBF

GeneChip Human Genome HGU133 Plus 2.0 Array

Complete coverage of the Human Genome U133 Set plus 6,500 additional genes for analysis of over 47,000 transcripts. The sequences from which these probe sets were derived were selected from GenBank®, dbEST, and RefSeq. The sequence clusters were created from the UniGene database (Build 133, April 20, 2001) and then refined by analysis and comparison with a number of other publicly available databases, including the Washington University EST trace repository and the University of California, Santa Cruz Golden-Path human genome database (April 2001 release).

In addition, there are 9,921 new probe sets representing approximately 6,500 new genes. These gene sequences were selected from GenBank, dbEST, and RefSeq. Sequence clusters were created from the UniGene database (Build 159, January 25, 2003) and refined by analysis and comparison with a number of other publicly available databases, including the Washington University EST trace repository and the NCBI human genome assembly (Build 31).

GeneChip Human Genome HGU133A 2.0 Array

The GeneChip® Human Genome U133A 2.0 Array is a single array representing 14,500 well-characterized human genes that can be used to explore human biology and disease processes. New design and reduced feature size mean that you can use smaller sample volumes than the previous HG®U133A Array without compromising performance.

  • Provides coverage of well-substantiated genes in the transcribed human genome on a single array
  • Analyzes the expression level of 18,400 transcripts and variants, including 14,500 well-characterized human genes
  • Comprised of more than 22,000 probe sets and 500,000 distinct oligonucleotide features

Affymetrix Whole-Transcript Expression Arrays

View a complete list of Affymetrix Whole Transcript Expression arrays that can be run in the CBF.

GeneChip Human Exon 1.0 ST Array

With approximately four probes per exon and roughly 40 probes per gene, the GeneChip® Human Exon 1.0 ST Array enables two complementary levels of analysis—gene expression and alternative splicing.

Multiple probes per exon enable "exon-level" analysis and allow you to distinguish between different isoforms of a gene. This exon-level analysis on a whole-genome scale opens the door to detecting specific alterations in exon usage that may play a central role in disease mechanism and etiology.

The second level is "gene-level" expression analysis, in which multiple probes on different exons are summarized into an expression value of all transcripts from the same gene.

Exon arrays provide the most comprehensive coverage of the genome, including empirically supported and predicted transcribed sequences, enabling the discovery of previously unidentified novel events.

GeneChip Human Gene 1.0 ST Array

The GeneChip® Human Gene 1.0 ST Array is the latest product in the family of Affymetrix expression arrays offering whole-transcript coverage. Each of the 28,869 genes is represented on the array by approximately 26 probes spread across the full length of the gene, providing a more complete and more accurate picture of gene expression than 3' based expression array designs. The small format of the array makes it a cost-effective expression profiling solution for new microarray users.

The Gene 1.0 ST Array uses a subset of probes from the Human Exon 1.0 ST Array and covers only well-annotated content. Like the Exon 1.0 ST Array, “gene-level” analysis of multiple probes on different exons is summarized into an expression value representing all transcripts from the same gene.

GeneChip Scanner 3000 7G

GeneChip Scanner 3000 7GThe GeneChip® Scanner 3000 7G is an extension of the GeneChip® Scanner 3000 (GCS 3000) series that allows you to scan next-generation higher-density arrays, including SNP arrays with up to 900,000 SNPs, tiling arrays for transcription and all-exon arrays for whole-genome analysis.

The GCS 3000 7G combines space-saving and other design improvements with speed, superior performance and lower scanner-to-scanner variation to dramatically improve throughput and efficiency in your genetic analysis. The GCS 3000 7G also provides enhanced auto-focus and more consistent scanner-to-scanner biological performance to improve data integrity and data sharing between researchers.

In addition the GCS 3000 7G has been designed to accommodate future advancements in GeneChip® probe array technology. Combined with the GeneChip® AutoLoader, the GCS 3000 7G provides sample tracking, temperature control and walk-away freedom from scanning.

GeneChip Fluidics Station 450

GeneChip Fluidics Station 450

The GeneChip® Fluidics Station 450 is used for the wash and stain operation of Affymetrix GeneChip® arrays. The instrument includes design advancements to provide improved ease of use and true walk-away freedom dramatically improving efficiency in your genetic analysis.

Frequently Asked Questions: RNA Expression by Affymetrix

  1. How much RNA is required for each assay?
  2. Sample Type Expression Array Type Target Prep Method *^Minimum Amount of Total RNA Required Minimum Concentration
    Fresh 3' IVT Ambion MessageAmp Premier Kit 500ng 166.67ng/ul
    Fresh 3' IVT NuGEN 50ng-100ng  
    Fresh Gene ST 1.0 Ambion WT Expression Kit 500ng 166.67ng/ul
    Fresh Gene ST 1.0 NuGen 50ng-100ng  
    Fresh Exon 1.0 ST Ambion WT Expression Kit 500ng 166.67ng/ul
    Fresh Exon 1.0 ST NuGen 50ng-100ng  
    FFPET 3' IVT NuGen 100ng 33.34ng/ul
    FFPET Gene ST 1.0 NuGen 100ng 33.34ng/ul
    FFPET Exon 1.0 ST NuGen 100ng 33.34ng/ul
    *Quality, Quantity, and Concentration subject to verification by the CBF
    ^ These are the minimal amounts specified in the respective protocols. We recommend you provide us with excess in order to run QC analysis and to repeat assays if necessary
  3. Where can I get a list of genes represented on the Affymetrix GeneChip® expression arrays?

    The sequences represented on all commercial GeneChip® expression arrays can be obtained from the Affymetrix website using the NetAffx™ Analysis Center or the annotation and sequence files.

    Annotation and sequence files contain the complete entries for all probe sets on the array, taken from the NetAffx Analysis Center. They are intended to be used primarily in spreadsheet applications and database programs (such as SQL databases). Interactive and batch queries can be performed in the NetAffx Analysis Center to find information for individual probe sets of interest.

    Annotation files are available for most Affymetrix GeneChip® Arrays. They are listed with other available support material on the various array support pages.

    Descriptions of Annotation Formats:

    MAGE-ML XML Annotations
    Manual, Probe Set Data in MAGE-ML Format

    CSV Annotations
    Manual, Probe Set Data in Tabular Format

    Alignments
    Manual, Alignments to Genome in PSL Format

  4. How can I find out if my genes of interest are on an Affymetrix GeneChip® expression array?

    The sequences represented on all commercial GeneChip® expression arrays can be obtained from the NetAffx™ Analysis Center.

    To search for a gene of interest, go to the Quick Query in the NetAffx Analysis Center. You will be required to log in with your email and password. Highlight the desired array type in #1 and enter the gene name, symbol, probe set ID, or public accession number in #2. Then, click on “Search.”

    To search the NetAffx Analysis Center using a list of genes, use the Batch Query option in the NetAffx Analysis Center. You will be required to log in with your email and password. For your genes of interest, create a tab-delimited text file in Excel or Notepad including gene symbols, probe set IDs, or Accession Numbers (in a column, excluding all header and footer information). Select the desired array type in #1, choose the appropriate search criteria in #2, and upload the .txt file in #3. Now click on “Search.”