TRANSCRIPTOME 2002: From Functional Genomics to Systems
A Robust DNA Based Amplification Technique for Tissue-Limiting Experiments on cDNA Microarrays
L. Smith1, P. Underhill1, C. Pritchard1, Z. Tymowska-Lalanne1, S. Abdul-Husein1, H. Hilton1, L. Winchester1, D. Williams2, T. Freeman2, and A. Greenfield1, 1Medical Research Council, Mammalian Genetics Unit, Harwell, Oxfordshire, UNITED KINGDOM, 2Medical research Council, UK Human Genome Mapping Project-Resource Centre, Hinxton, Cambridge, UNITED KINGDOM
AbstractThe potential of cDNA microarray analyses to address complex problems in a wide variety of fields is steadily being realised. The limiting factor in such analysis is often the amount of RNA required usually microgram quantities. To address this problem, researchers have turned to methods of improving detection sensitivity, either through increasing fluorescent signal output per molecule or increasing the amount of RNA through amplification. The key features of any amplification technique are that it is 1, reproducible, 2, reliable, 3, unbiased, 4, easy to use, and 5, cost effective. In vitro transcription of cDNA template has to date been the method of choice, however, this method has potential problems associated with RNA instability and degradation. Here we present a novel alternative method of amplification that increases the stability of target by using a DNA rather than RNA amplification procedure. The reproducibility, bias, and sensitivity of this technique has been examined using a 6000 EST microarray set and gives comparable results with as little as 30 ng total-RNA starting material.
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