title: Identification of reference genes for RT-qPCR in ovine mammary tissue during late pregnancy and lactation and in response to maternal nutritional programming.
abstract: 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.
authors.affiliation: 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.
authors.forename: A M
authors.initials: AM
authors.lastname: Paten
authors.affiliation: 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.
authors.forename: S J
authors.initials: SJ
authors.lastname: Pain
authors.affiliation: 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.
authors.forename: S W
authors.initials: SW
authors.lastname: Peterson
authors.affiliation: 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.
authors.forename: H T
authors.initials: HT
authors.lastname: Blair
authors.affiliation: 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.
authors.forename: P R
authors.initials: PR
authors.lastname: Kenyon
authors.affiliation: Laboratory for Evolution and Development, Genetics Otago, Department of Biochemistry, University of Otago, Dunedin, Aotearoa-New Zealand; Gravida: National Centre for Growth and Development, Aukland, New Zealand.
authors.forename: P K
authors.initials: PK
authors.lastname: Dearden
authors.affiliation: Laboratory for Evolution and Development, Genetics Otago, Department of Biochemistry, University of Otago, Dunedin, Aotearoa-New Zealand; Gravida: National Centre for Growth and Development, Aukland, New Zealand elizabeth.duncan@otago.ac.nz.
authors.forename: E J
authors.initials: EJ
authors.lastname: Duncan
mesh_terms: D000818:Animals; D019175:DNA Methylation; D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D007774:Lactation / Q000235:genetics*; D008321:Mammary Glands, Animal / Q000378:metabolism*; D039382:Maternal Nutritional Physiological Phenomena; D008957:Models, Genetic; D011247:Pregnancy; D011270:Pregnancy, Animal; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012756:Sheep; D034561:Sheep, Domestic / Q000235:genetics
keywords: RT-qPCR; mammary gland; nutritional programming; ovine; reference gene