Cai Jenny Hong, Deng Shibing, Kumpf Steven W, Lee Patricia A, Zagouras Panayiotis, Ryan Anne, Gallagher Dan S
Pfizer Global Research and Development, Pfizer Inc., Groton, CT 06340, USA.
Biotechniques. 2007 Apr;42(4):503-12. doi: 10.2144/000112400.
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.
实时定量聚合酶链反应（Real-time PCR）在基因表达谱研究中变得越来越重要，并且人们普遍认为，为了准确估计信使核糖核酸（mRNA）的表达，需要标准化数据。随着临床前研究和毒理基因组学中基因表达谱分析的增加，对大鼠内参基因的需求应运而生，而该领域的研究尚未得到充分评估。我们研究的目的是评估一组大鼠内参基因在不同组织类型中的基因表达变化。我们基于其假定的稳定性选择了48个已知的靶基因。使用TaqMan低密度阵列（LDA）技术在11种大鼠组织中检测了所有靶标的基因表达。使用两步统计模型评估每个基因的变异性。结果表明，使用多个内参基因分析平均表达可提供准确可靠的标准化表达数据。建议为未来的组织特异性研究从每种特定组织中选择至少五个变异性最小的基因。最后，推荐使用LDA平台对一组显示出最小变异的被研究大鼠内参基因进行进一步评估。我们的工作应能大大提高研究人员为给定实验简单有效地鉴定合适内参基因的能力。
