The Genomics Shared Resource provides centralized, cost-effective, and comprehensive expertise and service in the area of microarray-based and next-generation sequencing-based genomics research.

The following is helpful information for users, including array types available, researcher sample preparation, policies, useful protocols and data analysis tools.

Affymetrix GeneChips are oligonucleotide microarrays that provide a platform for full genome gene expression and genomic analysis. Affymetrix has developed this platform based on stringent probe-selection algorithms and a manufacturing technology that uses a combination of photolithography and solid-phase combinatorial chemistry.

The GSR currently runs Affymetrix GeneChips designed in an 11-micron feature-size format and an 18-micron feature-size format. The 18-micron format GeneChips have been in use at the GSR since early 2001. The 11-micron format GeneChips were introduced by Affymetrix in late 2003. These Chips are designed with increased probe density, allowing for whole-genome expression analysis on a single Chip.

The 11-micron format GeneChips currently stocked by the Genomics and Expression Resource are:

  • Human Genome U133 Plus 2.0 Array * —  This whole-genome array uses the new smaller feature-size technology that allows one GeneChip to contain all the probes from the U133A and U133B chips, plus 6,500 additional genes from GenBank, dbEST, and RefSeq.
  • Mouse Expression 430 2.0 Array † — This whole-genome array uses the new smaller feature-size technology that allows one GeneChip to contain all the probes from the 430A and 430B chips.

Other 11-micron arrays available are the HG-133A 2.0 array, Mouse Expression 430A 2.0 array, the Rat Genome 230 2.0 array, the Canine Genome array, the Chicken Genome array, the Drosophila 2.0 array, the Soy Genome array, and the V. vinifera Genome array. Upon researcher request, these GeneChips can be ordered by the GSR in the quantities required to accommodate a specific project.

The 18-micron format GeneChips used extensively in the Genomics and Expression Resource are:

  • Human Genome HG-U133A * — This array was released in January 2002 and is based on the draft sequence of the human genome. It contains a significant increase in the number of genes per array as compared to the U95Av2 and represents a majority of the 33,000 well-characterized human genes.
  • Human Genome Focus Array † — This array provides the researcher with a condensed array that represents about 8,500 highly annotated sequences in the NCBI RefSeq database. The probe sets contained in the Focus Array are derived directly from the Human Genome U133 Set, along with the normalization probe sets from that set. As a result, data from the Focus Array can be compared directly with data from the U133 set.
  • Mouse Expression 430A Array † — This array includes more than 34,000 well-characterized mouse genes. It is based on sequences in the UniGene database (build 107) and refined using the draft sequence of the mouse genome. It represents sequences from the NCBI RefSeq database and probe sets related to sequences from the MG-U74Av2 array.
  • Rat Expression 230A Array ‡ —  This array includes more than 28,000 well-characterized rat genes and based on rat genome sequences from the UniGene database (build 99), as well as sequence data from the Baylor rat genome draft assembly. This array incorporates sequences from the NCBI RefSeq database and sequences related to the probe sets on the RG-U34A array. 

Other arrays are available from Affymetrix and are described in the “Products” section of the Affymetrix website. These arrays will be purchased by the GSR upon request by the researcher. Please note that the researcher should allow for increased processing time and make prior arrangements with the GSR for expression analysis to be performed on chips not customarily stocked by the GSR.


* Relationship of the HG-133 Arrays to the Human Genome U95 Arrays.
The HG-U133A Array represents the vast majority of the genes featured on its predecessor, the HG-U95Av2 Array. In addition, a large percentage of the content on the HG-U95B, HG-U95C, HG-U95D, and HG-U95E arrays is represented on the HG-U133 Set. Due to the dynamic nature of the public sequence and gene databases, probe sets for these sequences will not be identical, and in some cases will be represented by a completely new probe set. As a result, data generated with different versions of the human arrays may not produce concordant results. Please call us for any questions.

† Relationship of the Mouse Expression 430 Arrays to the Murine Genome U74v2 Arrays.
Most sequences previously represented on the GeneChip Murine Genome U74Av2 Array are represented on the GeneChip Mouse Expression Array 430A. Due to the dynamic nature of public databases, probe sets for these sequences will not be identical, and in some cases will be represented by a completely new probe set. As a result, data generated with different versions of the mouse arrays may not always produce concordant results. Please call us for any questions.

‡ Most sequences previously represented on the GeneChip Rat Genome U34A Array are represented on the GeneChip Rat Expression Array 430A. Due to the dynamic nature of public databases, probe sets for these sequences will not be identical, and in some cases will be represented by a completely new probe set. As a result, data generated with different versions of the mouse arrays may not always produce concordant results. Please call us for any questions.

  • RNA gel image
  • Total RNA: For each sample submitted for analysis, the GSR requires high-quality total RNA, dissolved in RNase-free ultrapure water. The success of each gene expression experiment depends highly on the quality of the starting material. For this reason, the GSR recommends that stringent quality assessment measures be applied to the total RNA submitted for analysis. The following should be used as guidelines for total RNA submitted to the GSR for analysis:
    • RNA total quantity: In order to ensure that enough labeled cRNA is obtained for GeneChip hybridization, a minimum of 30 micrograms (30 µg) of total RNA is required per sample.
      • For the Small Sample GeneChip Analysis, a minimum of 200 nanograms of total RNA is required.
    • RNA concentration/volume: Total RNA should be submitted at a concentration of 0.3 µg/µL or higher. The total volume must be no more than 100 µL.
      • For the Small Sample GeneChip Analysis, total RNA should be submitted in a volume of no less than 10µL and no more than 100µL.
    • RNA integrity: The total RNA submitted to the GSR must be checked for RNA integrity. The predominant bands on the gel should be the 28S and 18S rRNA bands; (if you run these with a 1kb DNA marker, the 28S band will be at about 1500bp, and the 18S band will be at about 800bp). The relative intensities of these bands are a good indication of the RNA sample's integrity; the 28S band should be 1.5 to 2 times brighter than the 18S band, and there should be relatively little smearing in the gel. Please keep in mind that higher molecular weight material (especially in the gel's wells) may indicate DNA contamination and also may affect gene expression analysis.
    • A260 / A280 ratio: This is an index of nucleic acid purity. For best GeneChip results, the ratio of absorbances at λ =260 versus λ =280 should be between 1.8 and 2.0.
      • For the Small Sample GeneChip Analysis, if there is not sufficient sample to assess on a traditional spectrophotometer, the GSR can perform quantitation using our Nanodrop (for an additional fee).

The GSR strongly suggests that total RNA be extracted from cultured cells or tissue using the two-step TRIzol/acid phenol method described in our Genomics Shared Resource protocol or the Qiagen RNeasy kit. The two-step TRIzol/acid phenol method also can be used for isolation of RNA from whole blood, but there are alternative methods available that are more robust for use with gene expression arrays. If you are planning on performing a study on whole blood samples, please contact the GSR for advice and recommendations.

RNA Isolation:

These are the protocols that we have found work best with Affymetrix GeneChips. We do not recommend using other protocols to isolate RNA for submission to the GSR.

Useful RNA-related links:

There are a number of useful online resources related to working with RNA. Below are the links to some of them. If you have found others that you feel are useful, please feel free to let us know and we will post them.

Affymetrix protocols:

Free Tools and Software

  • dChip: Software package providing a model-based approach to probe-level analysis of Affymetrix GeneChips. Analysis features include: comparative analysis, hierarchical clustering, LDA, and plug-ins for ANOVA testing.
  • Bioconductor Software Packages: Open source and open development project to provide tools for analyzing and visualizing gene expression data via R software. Options include Robust Multi-Array Analysis (RMA) probe-level analysis plug-ins.
  • BRB ArrayTools: Microarray analysis tool utilizing a Microsoft Excel front-end. BRB ArrayTools provides visualization and statistical analysis tools in a familiar user-interface
  • NetAffx: Affymetrix-developed analysis center providing in-depth, highly curated probe set annotations to all users. Features include: array content searches, probe-target alignments, batch queries, UCSC Genomic Queries, SNP Finder, and the GeneOntology (GO) Mining Tool.
  • GenMAPP: Application enabling the integration of gene expression data with maps of biological pathways and ontology.
  • Onto-Express: Software tools enabling the translation of lists of differentially regulated genes into Gene Ontology groupings with a determination of statistical significance of each group.

Commercial Software

  • GeneSpring
  • Partek
  • Ingenuity Pathway Analysis