Validation of rat reference genes for improved quantitative gene expression analysis using low density arrays.

Real-time PCR has become increasingly important in gene expression profiling research, and it is widely agreed that normalized data are required for accurate estimates of messenger RNA (mRNA) expression. With increased gene expression profiling in preclinical research and toxicogenomics, a need for reference genes in the rat has emerged, and the studies in this area have not yet been thoroughly evaluated. The purpose of our study was to evaluate a panel of rat reference genes for variation of gene expression in different tissue types. We selected 48 known target genes based on their putative invariability. The gene expression of all targets was examined in 11 types of rat tissues using TaqMan low density array (LDA) technology. The variability of each gene was assessed using a two-step statistical model. The analysis of mean expression using multiple reference genes was shown to provide accurate and reliable normalized expression data. The least five variable genes from each specific tissue were recommended for future tissue-specific studies. Finally, a subset of investigated rat reference genes showing the least variation is recommended for further evaluation using the LDA platform. Our work should considerably enhance a researcher's ability to simply and efficiently identify appropriate reference genes for given experiments.

Molecular cytogenetic assignment of genes to bovine chromosome 5.

Seven genes were assigned by molecular cytogenetic methods to bovine chromosome 5. To accomplish this, specific primers were either publicly available or were designed from highly conserved regions of the publicly available mammalian gene sequences. The identity of the amplified segments was verified by sequencing and alignment with the published sequences. The optimized primers that amplified the desired bovine genes were used for screening a bovine bacterial artificial chromosome library. The positive clones were localized to a specific band of bovine chromosome 5 by fluorescence in situ hybridization. The genes HOXC4, SP1 and IGFBP6 were localized to band q21, COL2A1 was localized to bands q21-q23, IGF1 was localized to band q26, MB to band q31 and the gene CYP2D6 was localized to band q35. The cytogenetic assignment of SP1, IGFBP6, COL2A1, IGF1, MB and CYP2D6 is first reported here and the assignment of HOXC4 refines the previous assignment of this gene. The identification and localization of these genes further support the development of the human to bovine comparative map through characterizing the homologous segments conserved in the evolution of these species. This information will be useful for the future localization of genes that affect economically important traits in bovines.