title: Cross-platform microarray meta-analysis for the mouse jejunum selects novel reference genes with highly uniform levels of expression.
abstract: Reference genes (RGs) with uniform expression are used for normalization of reverse transcription quantitative PCR (RT-qPCR) data. Their optimization for a specific biological context, e.g. a specific tissue, has been increasingly considered. In this article, we compare RGs identified by expression data meta-analysis restricted to the context tissue, the jejunum of Mus musculus domesticus, i) to traditional RGs, ii) to expressed interspersed repeated DNA elements, and iii) to RGs identified by meta-analysis of expression data from diverse tissues and conditions. To select the set of candidate RGs, we developed a novel protocol for the cross-platform meta-analysis of microarray data. The expression stability of twenty-four putative RGs was analysed by RT-qPCR in at least 14 jejunum samples of the mouse strains C57Bl/6N, CD1, and OF1. Across strains, the levels of expression of the novel RGs Plekha7, Zfx, and Ube2v1 as well as of Oaz1 varied less than two-fold irrespective of genotype, sex or their combination. The gene set consisting of Plekha7 and Oaz1 showed superior expression stability analysed with the tool RefFinder. The novel RGs are functionally diverse. This facilitates expression studies over a wide range of conditions. The highly uniform expression of the optimized RGs in the jejunum points towards their involvement in tightly regulated pathways in this tissue. We also applied our novel protocol of cross-microarray platform meta-analysis to the identification of RGs in the duodenum, the ileum and the entire small intestine. The selection of RGs with improved expression stability in a specific biological context can reduce the number of RGs for the normalization step of RT-qPCR expression analysis, thus reducing the number of samples and experimental costs.
authors.affiliation: Genomics Core Facility, VetCore, University of Veterinary Medicine, Vienna, Austria.
authors.forename: Florian R L
authors.initials: FR
authors.lastname: Meyer
authors.forename: Heinrich
authors.initials: H
authors.lastname: Grausgruber
authors.forename: Claudia
authors.initials: C
authors.lastname: Binter
authors.forename: Georg E
authors.initials: GE
authors.lastname: Mair
authors.forename: Christian
authors.initials: C
authors.lastname: Guelly
authors.forename: Claus
authors.initials: C
authors.lastname: Vogl
authors.forename: Ralf
authors.initials: R
authors.lastname: Steinborn
mesh_terms: D000465:Algorithms; D000818:Animals; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D053263:Gene Regulatory Networks; D007583:Jejunum / Q000378:metabolism*; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D008297:Male; D015201:Meta-Analysis as Topic; D051379:Mice; D008810:Mice, Inbred C57BL; D008957:Models, Genetic; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D011506:Proteins / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D017422:Sequence Analysis, DNA; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics
chemical_list: D051741:Kruppel-Like Transcription Factors; D011506:Proteins; C031017:ornithine decarboxylase antizyme; C062076:zinc finger protein, X-linked; C524395:UBE2V1 protein, mouse; D044763:Ubiquitin-Conjugating Enzymes