Frequently Asked Questions

These are image files that can only be opened in the Feature Extraction Software. The jpg image can be opened in a variety of softwares.

If you choose to do your own analysis, we can help you with any supported software listed on out Data Analysis Page. (NEED LINK)

We can also perform data analysis for you.  Click here (LINK) for more information.

Duke investigators must purchase arrays directly from Affymetrix and bring them to the facility when submitting samples. Based on the volume of use, Duke investigators receive a considerable discount on the price of Affymetrix GeneChip® arrays. For more information on array pricing, or to determine the costs for probe synthesis, hybridization, and analysis of Affymetrix arrays, please contact us.

The Affymetrix GeneChip® system provides an approach to comparatively analyze genome wide patterns of gene expression using a technology that incorporates miniaturized, high density arrays of 25mer oligonucleotide probes. The probe arrays are manufactured by Affymetrix's proprietary, light directed chemical synthesis process, which generates high density arrays of oligonucleotides that possess a predefined position on the array. These arrays are used to monitor gene expression for thousands of transcripts.

There are several types of Affymetrix GeneChip® arrays. Our facility primarily works with three kinds: 3' expression arrays, Whole Transcript (Exon and Gene) arrays, and miRNA arrays.

1. 3' Expression arrays: these arrays have been a microarray platform standard for many years, with large quantities of published data available for comparison, and well developed functional annotations of the genes represented. Transcripts are represented as probe sets, where a probe set is made up of probe pairs comprised of perfect match (PM) and mismatch (MM) probe cells. This probe pairing strategy identifes and minimizes the effects of non specific hybridization and background signal. The intensities of each probe pair are used to determine the expression measurement. This measurement is calculated for each probe set and is described in the form of qualitative and quantitative values using the Affymetrix GeneChip® Command Console software, v.1.1. Array quality control metrics are well developed and understood for these arrays.

Target preparation from totalRNA uses T7 primers (incurring a 3' bias of transcript sensitivity) in the cDNA synthesis step, followed by in vitro transcription to produce biotin-labeled cRNA. The cRNA is fragmented and mixed into a hybridization cocktail before application to the array, which is then hybridized for 16 hours at 45C with rotation at 60rpm.

2. Whole Transcript (WT) arrays: these arrays have been developed more recently to enable analysis of the entire length of the genomic transcript, not merely the 3' end. This offers the advantage of more complete and precise expression information, and may provide new insights. However, there is not yet a wealth of published data avaialble for comparison and statistical analysis methods have not yet been standardized. Very little array quality control metrics are available.

  • Exon 1.0 ST arrays offer the most comprehensive analysis, with both alternative splicing information (exon-level analysis) and gene expression information that is derived from multiple probes on different exons and summarized into a single value for all transcripts from the same gene (gene-level analysis).
  • Gene 1.0 ST arrays are available for human, mouse, and rate, and offer a reduced WT approach that enables gene-level analysis for the well-annotated genes at a more affordable cost than the Exon 1.0 ST arrays.

Target preparation from totalRNA uses T7 primers in the cDNA synthesis step, followed by in vitro transcription to produce cRNA. Random primers are then used to synthesize cDNA from the cRNA. The cDNA is fragmented, biotin-labeled, and mixed into a hybriziation cocktail before application to the array, which is then hybrized for 17 hours at 45C with rotation at 60rpm.

3. miRNA arrays: released in 2009, these arrays provide expression analysis pioneers a tool to detect non-coding RNAs, including human, mouse, rat, canine, and monkey miRNAs, Sanger miRNA database V1.1 content and additional human small nucleolar RNAs (snoRNAs and scaRNAs). Very little data has been published, and few array quality control metrics are available.

Target preparation from totalRNA uses Genesphere® FlashTag Biotin Labeling kit. After poly-A tailing, the totalRNA is biotin-labeled in the FlashTag Ligation step, and mixed into a hybridization cocktail before application to the array. The array is hybridized for 16 hours at 48C with rotation at 60rpm.

Immediately following hybridization, the hybridized probe arrays undergo an automated washing and staining protocol on the GeneChip® FS450 fluidic station and are then scanned on the GeneChip® Scanner 3000 7G Plus where patterns of hybridization are detected. The hybridization data are collected as light emitted from the fluorescent reporter groups already incorporated into the target, which is now bound to the probe array. Probes that perfectly match the target generally produce stronger signals than those that have mismatches. Data generated from the scan is then analyzed using the Affymetrix GeneChip® Command Console software, v1.1, and the Affymetrix GeneChip® Expression Console software, v1.1. Affymetrix miRNA data is analysed using the miRNA QC Tool.

Feature Extraction is the process by which data is extracted from the scanned microarray image (.tif) and translated into log ratios, allowing researchers to measure DNA copy number changes in their experiments.

Agilent MicroArray Design Identifier

The first 6 digits of the design file indicate the AMADID. For example, if the design file is 012097_D_20070820, 012097 is AMADID and 20070820 is YYYYMMDD that the design file was generated.

Have I said thank you recently? You and Darren are being so helpful to all of us.

For each array experiment that is performed using Agilent arrays, four files with identical names but different extensions will be generated (*MAGEML xml, *txt, *pdf, *jpg).

For each array experiment that performed using the Affymetrix GeneChip® arrays, six files are generated (*.DAT, *.CEL, *.ARR, *RPT, *.txt).  Each file will be named identically except for the extension.  The naming convention is as follows:

Project ID number_Submission ID number_Genome ID number_samle name_chip type, i.e.  1234_0001_12123_CX24R_HU133+2.*

In the case of pairwise comparisons in the Affymetrix Microarray Suite v5.0, the comparison files will follow the following naming convention: Sample_base_Sample_exp.txt

All data analyses will be given as *.txt files.

The types of extensions are as follows:

ExtensionDescriptionComments
*.DATScanned image of the GeneChip arrayCan only be opened in Affymetrix
*.CELCell intensity file that calculates the average intensities for each cell and assigns it to an x,y cooridinate posiionCan only be opened in Affymetrix GeneChip Expression Console
*.CHPContains analysis outputCan be opened in Excel to manipulate
*.ARRContains experimental informationCan only be opened in Affymetrix
GeneChip Expression Console
*.RPTContains quality control information about the chipCan be opened in Excel to manipulate
*.txtContains analysis outputCan be opened in Excel to manipulate

Go to the Affymetrix website to view the various GeneChips® available. Go to "Tools & Data" under each chip to download genelists.

Only the sample of interest is necessary (a reference sample is not used).  Samle guideliens are as follows:

  1. 3' Expression Arrays: for complete processing, at least 200ng** of totalRNA in 8μl RNase-free water; for hybridization only from Affymetrix protocols, 20μg fragmented biotin-cRNA in 40μl volume (49 Format array) OR 15μg fragmented biotin-cRNA in 30μl volume (100 or 169 Format array) is required, according to the protocol; for hybridization only from NuGen WT-Ovation Pico protocol, 5μg fragmented and labeled cDNA in 50μl volume, according to the protocol.  **Note: At least 2μg in 8μl RNase-free water is required for the "old" Affymetrix One-Cycle IVT processing.
  2. WT Arrays: Exon-for complete processing, at least 1μg of totalRNA in 8μl RNase-free water; for hybridization only, 5.5μg fragmented and labeled ssDNA in 60ul volume, according to the protocol.  Gene-fore complete processing, at least 100ng of totalRNA in 8μl RNase-free water; for hybridization only, 5.5μg fragmented and labeled ssDNA in 60ul volume, according to the protocol.
  3. miRNA Arrays: for complete processing, at least 1μg of totalRNA in 8μl RNase-free water.

For procedures starting from totalRNA we also perform an RNA quality check, so please bring an extra 3μl aliquot of your samle in a seperate tube.