title: Identification of reliable reference genes for qRT-PCR studies of the developing mouse mammary gland.
abstract: Cell growth and differentiation are often driven by subtle changes in gene expression. Many challenges still exist in detecting these changes, particularly in the context of a complex, developing tissue. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) allows relatively high-throughput evaluation of multiple genes and developmental time points. Proper quantification of gene expression levels by qRT-PCR requires normalization to one or more reference genes. Traditionally, these genes have been selected based on their presumed "housekeeping" function, with the implicit assumption that they are stably expressed over the entire experimental set. However, this is rarely tested empirically. Here we describe the identification of novel reference genes for the mouse mammary gland based on their stable expression in published microarray datasets. We compared eight novel candidate reference genes (Arpc3, Clock, Ctbp1, Phf7, Prdx1, Sugp2, Taf11 and Usp7) to eight traditional ones (18S, Actb, Gapdh, Hmbs, Hprt, Rpl13a, Sdha and Tbp) and analysed all genes for stable expression in the mouse mammary gland from pre-puberty to adulthood using four different algorithms (GeNorm, DeltaCt, BestKeeper and NormFinder). Prdx1, Phf7 and Ctbp1 were validated as novel and reliable, tissue-specific reference genes that outperform traditional reference genes in qRT-PCR studies of postnatal mammary gland development.
authors.affiliation: Section of Molecular Cytology and Van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands.
authors.forename: Anoeska Agatha Alida
authors.initials: AA
authors.lastname: van de Moosdijk
authors.affiliation: Section of Molecular Cytology and Van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands.
authors.forename: Renée
authors.initials: R
authors.lastname: van Amerongen
mesh_terms: D000328:Adult; D000429:Alcohol Oxidoreductases / Q000235:genetics*; D000465:Algorithms; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D066264:Datasets as Topic; D005260:Female; D018507:Gene Expression Regulation, Developmental; D056726:Genetic Association Studies; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008321:Mammary Glands, Animal / Q000502:physiology*; D051379:Mice; D008815:Mice, Inbred Strains; D046228:Microarray Analysis; D009928:Organ Specificity; D038081:Organogenesis / Q000235:genetics; D054464:Peroxiredoxins / Q000235:genetics*; D011627:Puberty; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D004268:DNA-Binding Proteins; D000429:Alcohol Oxidoreductases; C096794:C-terminal binding protein; D054464:Peroxiredoxins; C489899:Prdx1 protein, mouse