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microarray history
In February 2001, two independent research groups published their initial drafts of the human genome sequence (1,2). This is widely considered to be one of the most significant events in the history of biology. In addition to the human genome, there are more than one hundred organisms for which the genome has been fully sequenced (3). For these organisms, focus has shifted to determining the functional information about the genes. This new focus has created a field of research known as Functional Genomics. The mass sequencing projects were analogous to identifying all the words in a language, whereas Functional Genomics ascribes meanings or definitions to the words, as well as uncovering the syntax of the language.
Functional Genomics projects are by their nature very large scale. They are also typically multidisciplinary, involving molecular biology, physics, chemistry and engineering. Robotics has been employed to allow for higher throughput analyses to be conducted. One approach to functional genomics involves identifying changes in the expression of genes under specific conditions. Many diseases for example are the result of an improper regulation of gene expression. Microarray technology, which allows for the simultaneous monitoring of expression from thousands of genes, has proven to be one of the most successful realisations of functional genomics approaches. The applications of microarrays extend beyond the boundaries of basic biology into diagnostics, environmental monitoring and pharmacology. In addition, pharmaceutical companies are using microarrays in order to expedite the drug discovery process.
The UHN Microarray Centre was initially established to ensure that Canadian scientists have access to high quality microarrays, at an affordable price, and access to technical and professional support. Now, the Microarray Centre distributes arrays to over 700 labs around the world.
Macroarrays to Microarrays
Microarrays evolved as scientists faced the challenge of finding high-throughput techniques that could be used to detect and determine the relative abundance of transcripts in RNA samples. In the early 1990’s, when microarrays were in their infancy, colonies were hand-printed onto nylon membranes with a 96-pin tool. These “macroarrays” were then hybridised with radioactively labelled samples to perform “1-colour” assays.
In the mid to late 1990’s, fluorescent labelling protocols grew in popularity. Although this labelling method required sophisticated instrumentation to detect the fluorescent signal, it allowed for 2-colour hybridisation assays(4-8). By enabling researchers to compare two different samples on the same array, expression profiles could be determined more reliably. In 1995, Schena et al. developed a high-capacity system to monitor the expression of 45 Arabidopsis genes in parallel by spotting complementary DNA (cDNA) onto microscope-sized glass slides using high-speed robotic printing (5). In this study, the miniaturised high-density format allowed for the use of smaller hybridisation volumes which improved the sensitivity of the assay and enabled detection of rare transcripts derived from 2 micrograms to messenger RNA (mRNA) (5). In 1996, Schena et al. reported the detection limit of the microarray assay allows profiling of transcripts that represent 1:500,000 (wt/wt) of the mRNA (4). While this was an improvement in sensitivity over the colony filters and radioactively labelled probe methods (which could quantify expression levels for transcripts present at 0.005% abundance in the sample (9)), it was far from the detection limits of today. Current protocols require as little as 2-3 micrograms of total RNA for direct or indirect (amino allyl) labelling. In addition, a number of RNA amplification techniques, both linear and exponential, have been developed to allow for transcript profiling on small samples, such as samples isolated by laser capture microdissection.
The microarray platform has benefited from a number of advancements over the past decade. Besides the evolution from hand-printing colonies onto nylon membranes to photolithographic printing of over 1.3 million features onto a glass slide, microarray production has also benefited from improvements to the proprietary coatings of the glass slides which yield better spot quality (consistent size and morphology). The development of many different fluorophores have allowed for multi-colour hybridisation assays using various conjugation chemistries and state-of-the-art instrumentation that has allowed for higher resolution scanning and higher-density printing. In terms of data management, software has been developed to enable researchers to easily archive, view, and statistically analyse a number of large data sets easily.
In addition to the simultaneous determination of the relative abundance of transcripts in two RNA samples, the microarray platform has expanded to include high-throughput mini-sequencing, SNP detection, array comparative hybridisation genomics (aCGH), differential methylation hybridisation (DMH), and chromatin immunoprecipitation on microarrays (ChIP-on-chip). As well, microarray platforms now encompass protein-related applications such as antibody arrays and cellular applications such as cell-based microarrays and tissue microarrays.
1. Venter,J.C., Adams,M.D., Myers,E.W., Li,P.W., Mural,R.J., Sutton,G.G., Smith,H.O., Yandell,M., Evans,C.A., Holt,R.A. et al. (2001) The sequence of the human genome. Science, 291, 1304-1351
2. THE GENOME INTERNATIONAL SEQUENCING CONSORTIUM (2001) Initial sequencing and analysis of the human genome. Nature 409, 860-921
3. Frazier, M.E., Johnston, G.M., Tomassen, D.G., Oliver,C.E., Patrinos, A. (2003) Realizing the Potential of the Genome Revolution: The Genomes to Life Program,Science. 300:290-293
4. Schena, M., et al., Parallel human genome analysis: microarray-based expression monitoring of 1000 genes. (1996) Proceedings of the National Academy of Sciences of the United States of America. 93(20): p. 10614-9.
5. Schena, M., Shalon, D., Davis, R.W., Brown, P.O. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science, 1995, 270(5235):467-470.
6. Shalon, D., Smith, S.J., Brown, P.O. A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization. Genome Research, 1996, 6:639-645.
7. Yang, G.P. et al. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes. Nucleic Acids Research, 1999, 27(6):1517-1523.
8. Diehn, M., Eisen, M.B., Botstein, D., Brown, P.O. Large-scale identification of secreted and membrane-associated gene products using DNA microarrays. Nature Genetics, 2000, 25:58-62.
9. Bernard, K, Auphan, N., Granjeaud, S., Victorero, G., Schmitt-Verhulst, A.-M., Jordan, B.R., Nguyen, C. Multiplex messenger assay: simultaneous, quantitative measurement of expression of many genes in the context of T cell activation. Nucleic Acids Research, 1996, 24(8):1435-1442.