Tissue Gene Expression Analysis Using Arrayed Normalized cDNA Libraries

TL;DR: Oligonucleotide-fingerprinting data on 60,000 cDNA clones from two different mouse embryonic stages is used to establish a normalized cDNA clone set and the identification of tissue-specific expression profiles and the detection of genes specifically expressed in the tissues investigated.

Abstract: The level of expression of all genes of an organism in different cell types, tissues, stages of development, or disease processes constitutes essential information for understanding the function of different genes and to unravel the complex network of biological processes acting in every biological system. A number of various approaches have been developed to gain information on gene expression levels, based either on counting the number of clones in libraries prepared from different materials or on some (typically hybridization) intensity measurements. Examples of this first approach are EST sequencing (Adams et al. 1993; Boguski and Schuler 1995), oligonucleotide fingerprinting (Meier-Ewert et al. 1998), or SAGE (Velculescu et al. 1995; Zhang et al. 1997). Complex cDNA hybridization (Lehrach et al. 1990; Lennon and Lehrach 1991; Gress et al. 1996; Duggan et al. 1999), whole-mount in situ hybridization (Wilkinson and Nieto 1993), and differential display technology (Liang and Pardee 1992) logically belong to the second class of approaches for analyzing patterns of gene expression. Among these techniques, the use of complex cDNA hybridization combined with high-density arrays of spotted cDNA clones, PCR products (Gress et al. 1992; Schena et al. 1995; DeRisi et al. 1996; Lashkari et al. 1997) or oligonucleotides (Southern 1995; Hoheisel 1997; Lipshutz et al. 1999), offers a number of advantages compared to many of the other approaches. This approach combines high sensitivity with a high throughput because of the possibility of an enormous number of parallel experiments carried out on a single high-density DNA array (Poustka et al. 1986; Lehrach et al. 1990; Schena et al 1996; Brown and Botstein 1999). To extract significant biological information from complex cDNA hybridization, computational analysis and clustering methods are necessary, providing an efficient technique to group together differentially expressed and functionally related genes (Eisen et al. 1998; Iyer et al. 1999). We have used oligonucleotide fingerprinting to establish a normalized subset of genes expressed during day 9 and 12 of mouse embryonic development comprising 5,376 cDNA clones. We used this selected set of cDNA clones to construct high-density cDNA arrays of PCR products spotted on glass surfaces, which then have been used in complex cDNA hybridization experiments. This approach of constructing a normalized clone set has particular advantages in less well characterized genomes, where neither a genomic sequence nor a precharacterized 'unigene' clone set is available (Lehrach et al. 1990; Meier-Ewert et al. 1998; Poustka et al. 1999). The selected set was used to determine tissue-specific gene expression profiles combining complex cDNA hybridization with statistical analysis.