Assessment of a systematic expression profiling approach in ENU-induced mouse mutant lines

Abstract

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.

Identification of coexpressed gene clusters in a comparative analysis of transcriptome and proteome in mouse tissues

“…The concentration of total RNA was measured by OD 260/280 reading. Per DNA chip, 20 g of total RNA were used for reverse transcription and indirectly labeled with Cy3 or Cy5 fluorescent dyes according to The Institute for Genomic Research (TIGR) protocol as described (19). Probes were PCR amplified from the 20,000 (20k) mouse arrayTAG clone set as described (20).…”

“…Amplified probes were dissolved in 3ϫ SSC and spotted on aldehyde-coated slides (CEL Associates, Pearland, TX) by using the Microgrid TAS II spotter (Biorobotics Genomic Solutions, Huntingdon, U.K.) with Stealth SMP3 pins (Telechem, Sunnyvale, CA). Spotted slides were rehydrated, blocked, denatured, and dried as described (19,21). The hybridization mixture was placed on prehybridized microarrays and hybridized at 42°C for 18-20 h. Microarrays were immersed in 40 ml of 3ϫ SSC and then successively washed in 40 ml of 1ϫ SSC, 40 ml of 1ϫ SSC͞0.1% SDS, and 40 ml of 0.1ϫ SSC at room temperature as described (19).…”

“…Spotted slides were rehydrated, blocked, denatured, and dried as described (19,21). The hybridization mixture was placed on prehybridized microarrays and hybridized at 42°C for 18-20 h. Microarrays were immersed in 40 ml of 3ϫ SSC and then successively washed in 40 ml of 1ϫ SSC, 40 ml of 1ϫ SSC͞0.1% SDS, and 40 ml of 0.1ϫ SSC at room temperature as described (19). Dried slides were scanned with a GenePix 4000A scanner and analyzed by using the GENEPIX PRO 3.0 image processing software (Axon Instruments, Union City, CA).…”

“…To analyze the differential transcriptome of mouse liver and kidney, RNA expression profiling was performed with cDNA microarrays (22) containing a sequence-verified 20,200 mouse clone set (19,21). Sixteen dual-color DNA chip hybridizations of cDNAs from age-matched C3HeB͞FeJ male mice were made (Table 1, chips 1 a-f, 2 a-f, and 3 a-d, which is published as supporting information on the PNAS web site).…”

Episcopic 3D Imaging Methods: Tools for Researching Gene Function

“…Tissue samples of normal or diseased model organisms or humans can be analyzed with the aid of microarray techniques [1][2][3][4][5][6]. Microarray studies provide information about the activity of thousands of genes in very short times.…”