No abstract

The cDNA-chip technology is a highly versatile tool for the comprehensive analysis of gene expression at the transcript level. Although it has been applied successfully in expression profiling projects, there is an ongoing dispute concerning the quality of such expression data. The latter critically depends on the specificity of hybridisation. SAFE (specificity assessment from fractionation experiments) is a novel method to discriminate between non- specific cross-hybridisation and specific signals. We applied in situ fractionation of hybridised target on DNA-chips by means of repeated washes with increasing stringencies. Different fractions of hybridised target are washed off at defined stringencies and the collected fluorescence intensity data at each step comprise the fractionation curve. Based on characteristic features of the fractionation curve, unreliable data can be filtered and eliminated from subsequent analyses. The approach described here provides a novel experimental tool to identify probes that produce specific hybridisation signals in DNA-chip expression profiling approaches. The iterative use of the SAFE procedure will result in increasingly reliable sets of probes for microarray experiments and significantly improve the overall efficiency and reliability of RNA expression profiling data from DNA-chip experiments.

Comparative genomewide expression profiling is a powerful tool in the effort to annotate the mouse genome with biological function. The systematic analysis of RNA expression data of mouse lines from the Munich ENU mutagenesis screen might support the understanding of the molecular biology of such mutants and provide new insights into mammalian gene function. In a direct comparison of DNA microarray experiments of individual versus pooled RNA samples of organs from ENU-induced mouse mutants, we provide evidence that individual RNA samples may outperform pools in some aspects. Genes with high biological variability in their expression levels (noisy genes) are identified as false positives in pooled samples. Evidence suggests that highly stringent housing conditions and standardized procedures for the isolation of organs significantly reduce biological variability in gene expression profiling experiments. Data on wild-type individuals demonstrate the positive effect of controlling variables such as social status, food intake before organ sampling, and stress with regard to reproducibility of gene expression patterns. Analyses of several organs from various ENU-induced mutant lines in general show low numbers of differentially expressed genes. We demonstrate the feasibility to detect transcriptionally affected organs employing RNA expression profiling as a tool for molecular phenotyping.

Single-copy sequences of chromosome bands 1q23 →1q25 were enriched by subtractive hybridization of digoxigenin oligonucleotide-labeled DNA from a somatic cell hybrid containing human chromosome 1 with DNA from a somatic cell hybrid containing a deleted human chromosome 1 del(l)(q23q25). These sequences were purified with an immobilized anti-digoxigenin antibody, amplified by polymerase chain reaction (PCR) and cloned. The majority of clones contained single-copy sequences, thus allowing direct screening of human genomic DNA. With pools of probes complex hybridization patterns as well as differences in hybridization patterns between genomes were observed in Southern blotting experiments. Nine clones containing independent single-copy sequences from chromosome bands 1q23→1q25 were isolated and sequenced to generate sequence-tagged sites (STSs). Using PCR, region specific genomic clones containing large inserts were identified in human genomic lambda phage and cosmid libraries. Thus, region specific genomic probes generated by genomic difference cloning are useful for identifying deletions and rearrangements, for generating STSs and for isolating genomic clones containing large inserts of a particular chromosomal region.