Microarray FAQs

Q: What is the standard workflow for a gene expression experiment?
A: After an initial meeting to discuss the project goals, experimental design, and logistics, our users deliver isolated totalRNA samples for each of their experimental conditions. We then check the RNA quality and concentration in the Agilent 2100 BioAnalyzer prior to ordering the expression arrays. If everything checks out, we proceed with the amplification, fragmentation, and labeling reactions. Next, a spectrophotometer reading on each sample tells us whether or not the amplification was successful, and if so, the samples are hybridized to the array. Following the overnight hybridization the arrays are scanned, and the raw data extracted. Upon completion of the experiment, the raw expression data is made available to our users, or we will assist them with the analysis.

Note: In the rare event that any of aforementioned reactions fail either fully or partially, users are contacted in order to resolve the issues before proceeding with the hybridization.

Q: I'm interested in studying gene expression in my samples. Which Affymetrix arrays should I be considering?
A: Expression arrays are available for the majority of model organisms. The Affymetrix 3' IVT expression arrays have become a well-established and trusted method in gene expression profiling.

Some of the most popular 3' IVT arrays are:
• Human Genome U133 Plus 2.0
• GeneChip Mouse Genome 430 2.0
• GeneChip Rat Genome 230 2.0
• GeneChip Drosophila Genome 2.0

More recent was the release of the whole-transcript expression arrays. Aimed at being a cheaper alternative to the traditional 3' IVT arrays, these are of a smaller format and contain only known, well-annotated genes. The figure below illustrates the differences in probe placement between the two array types.

A screenshot of the UCSC Genome browser page for human gene ATP8B2. Notice the greater distribution of probes in the exon array track (Affy HuEx 1.0) relative to that of the 3' IVT array track (Affy U133). Click on the image for a larger view.

These arrays are currently available for only the human, mouse and rat genomes:
• GeneChip Human Gene 1.0 ST
• GeneChip Mouse Gene 1.0 ST
• GeneChip Rat Gene 1.0 ST

Q. Does Affymetrix offer arrays for studying gene regulation?
A: Affymetrix Whole Genome Tiling Arrays may be employed to study the DNA sequences bound by regulatory proteins, as well as to identify sites of histone and DNA modification. In this particular type of array, probes are tiled at an average resolution of 35 base pairs as measured from the central position of adjacent 25-mer oligos, leaving a gap of approximately 10bp between probes.

For more information on some of the tiling arrays available, refer to the following pages:
• GeneChip® Human Tiling 2.0R Array Set
• GeneChip® Mouse Tiling 2.0R Array Set

In addition, species specific Promoter GeneChips are also available. Similar to the tiling arrays, probes are tiled at an average resolution of 35 base pairs as measured from the central position of adjacent 25-mer oligos, leaving a gap of approximately 10 base pairs between probes. Each promoter region covers approximately 6 kb upstream through 2.5 kb downstream of 5' transcription start sites.

For more information on the promoter arrays, refer to the following pages:
• GeneChip® Human Promoter 1.0R Array
• GeneChip® Mouse Promoter 1.0R Array

For a more comprehensive and up to date list of arrays and associated supporting material, please visit the the Affymetrix GeneChips website.

Q: How much RNA do I need to provide?
A: For 3' IVT arrays, we would like to see no less than 5ug of total RNA as a starting point. The RNA should be suspended at a concentration of at least 200 ng/ul, while higher concentrations will likely yield a clearer result.

The smaller format of the Gene 1.0 ST array means a decreased amount of required starting material. For these arrays, we would like to see no less than 200ng of RNA at a concentration of at least 67ng/ul.

Regardless of array type, keep in mind that 1ul of your sample will be used for QC purposes in the BioAnalyzer.

Q: Which RNA isolation protocol should I use?
A: The Affymetrix protocol suggests the following:
• Total RNA from Mammalian Cells: RNeasy Mini Kit from QIAGEN
• Total RNA from Mammalian Tissue: Use a commercial reagent, such as TRIzol

Q: Should my experiment include biological or technical replicates?
A: In order to derive reliable statistical measures from the experiment, we recommend running at least 3 samples per experimental condition. Technical replication is not necessary due to the level of standardization built in to Affymetrix technology.

Q: Will you be able to assist me with the data analysis?
A: Yes, we would be happy to assist you with the analysis of your microarray data. Most analyses are accomplished using a suite of R packages known as BioConductor. More information can be found at r-project.org or BioConductor.org. Please visit our list of recommended software to explore other analysis options.

Q: How much time will my experiment take?
A: It will normally take 2 - 3 weeks from the time we receive your samples before we see experimental results.