Development of Methods in the Field of Gene Expression Profiling

For the generation of comprehensive cDNA microarrays, large collections of E. coli clones are needed comprising tens of thousands of clones each of them containing one “foreign” gene specific cloned cDNA fragment, e. g., human gene specific fragment. These collections, available by respective clone banks (e. g., Deutsches Ressourcenzentrum Berlin, RZPD), will represent almost the complete set of genes or transcripts of an organism. In our group mouse and human specific collections are available. Using these large clone collections for the production of cDNA microarrays, we had first to advance our established protocols. By systematic methodological optimization of parameters, e. g., type and surface chemistry of glass surfaces, spotting buffer composition, minimization of background fluorescence intensity and maximization of spotting density, and, in addition, standardization of production conditions we could establish a powerful protocol which in principle allows us spotting of up to 70,000 gene specific probes per glass slide [Kokocinski et al., 2003; Wrobel et al., 2003].

When analysing tumor transcriptomes by DNA-microarray techniques, limitations of the amount of available RNA are a further strong problem, which can seriously impair or even circumvent the successful analysis of these tumors. Especially gene expression analysis using microarrays of synthetic long oligonucleotides is limited in that it requires substantial amounts of RNA. To obtain these quantities from minute amounts of starting material, protocols were developed that linearly amplify mRNA by cDNA synthesis and in vitro transcription. Since orientation of the product is antisense (aRNA), it is inapplicable for dye-labelling by reverse transcription and hybridisation to sense-oriented oligonucleotide arrays.
Therefore, we introduced a novel protocol [Schlingemann et al., 2005] in which aRNA labelling is achieved by a combination of two reverse and one forward transcription reactions followed by dye-incorporation using Klenow fragment, generating fluorescent antisense cDNA (see Figure 1). We demonstrated high fidelity in arrays using up to 105-fold amplification, starting from 2 ng total RNA. The generated data are highly reproducible and maintain relative gene expression levels between samples (see Figure 2). Our protocol is an efficient and reliable technique to expand the applicability of oligonucleotide arrays to studies where RNA is limited source material.