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Gene Expression Arrays:,,,Highly Sensitive Detection of Expression Patterns,,,with Improved Tools for Target Amplification

Introduction

As the human genome project is completed, microarray technology offers the potential to study the genomes complexity. This technology facilitates the direct extraction of functional information from nucleic acids by measuring the RNA levels of a complete organism or its associated subsets.

After isolation of total RNA, various methods can be applied to prepare targets for microarray screening (Figure 1). The most common procedure involves direct cDNA labeling by reverse transcription in which fluorescencelabeled nucleotides are incorporated. A clear limitation of this technology is the large amount of RNA required per hybridization. Some of the most important applications in medical research involve studying very small amounts of tissues (e.g., microdissected tissue [LCM] or biopsy material from tumors). Therefore, target amplification methods have been developed to overcome this limitation:

    The linear cRNA amplification procedure, based on reverse transcription with an oligodT primer connected to a T7 promoter and in vitro transcription of resulting DNA with T7 RNA polymerase [1, 2], amplifies a RNA target approximately 100-fold. This method does not significantly distort the relative abundance of individual mRNA sequences within an RNA population [3, 4].
    A PCR amplification method based on reverse transcription, followed by random PCR amplification of the cDNA and in vitro transcription of the resulting PCR product with T7 RNA polymerase, is illustrated in Figure 2. With this procedure as little as 50 ng total RNA (from 1,000 cells or 0.1 mg of
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Edited by I.H. Pawlowitzki, J.H. Edwards and E.A. Thompson (1997) • This volume covers: Linkage in dominant recessive and oligogenic disease, Model free (non-parametric) methods, Tools for gene ...
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