Paten A M, Pain S J, Peterson S W, Blair H T, Kenyon P R, Dearden P K, Duncan E J
Laboratory for Evolution and Development, Genetics Otago, Department of Biochemistry, University of Otago, Dunedin, Aotearoa-New Zealand; International Sheep Research Centre, Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, Aotearoa-New Zealand; and Gravida: National Centre for Growth and Development, Aukland, New Zealand.
International Sheep Research Centre, Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, Aotearoa-New Zealand; and Gravida: National Centre for Growth and Development, Aukland, New Zealand.
Physiol Genomics. 2014 Aug 1;46(15):560-70. doi: 10.1152/physiolgenomics.00030.2014. Epub 2014 Jun 3.
The mammary gland is a complex tissue consisting of multiple cell types which, over the lifetime of an animal, go through repeated cycles of development associated with pregnancy, lactation and involution. The mammary gland is also known to be sensitive to maternal programming by environmental stimuli such as nutrition. The molecular basis of these adaptations is of significant interest, but requires robust methods to measure gene expression. Reverse-transcription quantitative PCR (RT-qPCR) is commonly used to measure gene expression, and is currently the method of choice for validating genome-wide expression studies. RT-qPCR requires the selection of reference genes that are stably expressed over physiological states and treatments. In this study we identify suitable reference genes to normalize RT-qPCR data for the ovine mammary gland in two physiological states; late pregnancy and lactation. Biopsies were collected from offspring of ewes that had been subjected to different nutritional paradigms during pregnancy to examine effects of maternal programming on the mammary gland of the offspring. We evaluated eight candidate reference genes and found that two reference genes (PRPF3 and CUL1) are required for normalising RT-qPCR data from pooled RNA samples, but five reference genes are required for analyzing gene expression in individual animals (SENP2, EIF6, MRPL39, ATP1A1, CUL1). Using these stable reference genes, we showed that TET1, a key regulator of DNA methylation, is responsive to maternal programming and physiological state. The identification of these novel reference genes will be of utility to future studies of gene expression in the ovine mammary gland.
乳腺是一种复杂的组织，由多种细胞类型组成，在动物的一生中，会经历与怀孕、哺乳和退化相关的反复发育周期。乳腺也已知对营养等环境刺激引起的母体编程敏感。这些适应性变化的分子基础备受关注，但需要可靠的方法来测量基因表达。逆转录定量PCR（RT-qPCR）常用于测量基因表达，目前是验证全基因组表达研究的首选方法。RT-qPCR需要选择在生理状态和处理过程中稳定表达的参考基因。在本研究中，我们确定了适合的参考基因，用于对绵羊乳腺在两种生理状态（妊娠后期和哺乳期）下的RT-qPCR数据进行标准化。从孕期经历不同营养模式的母羊所产后代中采集活检样本，以检查母体编程对后代乳腺的影响。我们评估了八个候选参考基因，发现对来自混合RNA样本的RT-qPCR数据进行标准化需要两个参考基因（PRPF3和CUL1），但分析个体动物的基因表达需要五个参考基因（SENP2、EIF6、MRPL39、ATP1A1、CUL1）。使用这些稳定的参考基因，我们表明DNA甲基化的关键调节因子TET1对母体编程和生理状态有反应。这些新参考基因的鉴定将对未来绵羊乳腺基因表达研究有用。
