GeneChips from Affymetrix comprise a whole family of microarrays for genome-wide gene expression monitoring studies in mammalian species as well as in non-vertebrates. In these arrays, the so called oligonucleotide type microarrays, each gene is represented by multiple probe pairs with a bias toward the 3`end of the gene. Each probe pair consists of a perfect match and a mismatch oligonucleotide (that is, on average, 20 base pairs in length). The mismatch oligonucleotide contains a single base pair mismatch in the center of the probe. The sequence of the probes is chosen to be specific for a particular gene and the probe pairing design facilitates identification and subtraction of non-specific hybridization and background signals. Murine GeneChip consists of three arrays, U74A, -B and –C. The U74A contains oligonucleotide probes specific for 6,000 known mouse genes, present in the Mouse UniGene database, and 6,000 EST sequences. Arrays –B and –C contain, in total, 24,000 EST sequences. The Human GeneChip comprises five U95 arrays from U95A to –E. All of them, except the A array, contain solely EST sequences (50,000). The U95A array contains 12,000 full-length human genes, previously characterized functionally and in terms of the disease association.
The preparation of the mRNA used to query the arrays is fairly straightforward; the mRNA is reverse transcribed into cDNA, this cDNA is then used for an in vitro transcription reaction that yields biotin-labeled cRNA. The biotin-labeled cRNA is then hybridized to the arrays. Hybridization signals are developed using immunochemistry and detected using a high-resolution, Hewlett Packard scanner as a part of the Affymetrix GeneChip Instrument System.
We used the U74A GeneChip to investigate genome-wide expression profiles in different subpopulations of murine T helper cells. The U95A arrays helped us to elucidate gene expression differences in particular subpopulations of human B cell precursors. In both projects we aimed to screen only known genes, therefore we used the A series in both types of chips. In general, we were very satisfied with the results obtained. The real advantage of the Affymetrix microarrays is that the technology is already well established. The probe preparation and hybridization protocols are clearly written and the obtained experimental results are reproducible. Compared to other companies active in the molecular biology business, technical help from Affymetrix is particularly competent and effective. Also, the match/mismatch probe system improves the sensitivity of analysis. The presence of reference genes is a useful feature for the normalization and quantitation of the data from different experments.
There are some issues on GeneChips, however, that have to be improved. The bias toward 3‘ end of the targed gene, which is inherited in probe selection does not allow a detection of alternatively spliced transcripts. Unfortunately, GeneChips cannot be re-used, which significantly raises the costs of experiments. For those planning to use chips from Affymetrix I would also strongly suggest using chips from the same batch, since we observed up to 3% differences in the performance of chips from different deliveries.
Taken together I would recommend a purchase of GeneChips for those researchers who wish to obtain a broad spectrum of gene expression data in a reasonably short time. These arrays allow one to avoid time-consuming optimization steps making them a quick way to generate gene expression profiles.
Michal Janitz, M.D.
Groupleader
Max Planck Institute for Molecular Genetics
Berlin, Germany
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