title: Identification of internal control genes for quantitative expression analysis by real-time PCR in bovine peripheral lymphocytes.
abstract: Gene expression studies in blood cells, particularly lymphocytes, are useful for monitoring potential exposure to toxicants or environmental pollutants in humans and livestock species. Quantitative PCR is the method of choice for obtaining accurate quantification of mRNA transcripts although variations in the amount of starting material, enzymatic efficiency, and the presence of inhibitors can lead to evaluation errors. As a result, normalization of data is of crucial importance. The most common approach is the use of endogenous reference genes as an internal control, whose expression should ideally not vary among individuals and under different experimental conditions. The accurate selection of reference genes is therefore an important step in interpreting quantitative PCR studies. Since no systematic investigation in bovine lymphocytes has been performed, the aim of the present study was to assess the expression stability of seven candidate reference genes in circulating lymphocytes collected from 15 dairy cows. Following the characterization by flow cytometric analysis of the cell populations obtained from blood through a density gradient procedure, three popular softwares were used to evaluate the gene expression data. The results showed that two genes are sufficient for normalization of quantitative PCR studies in cattle lymphocytes and that YWAHZ, S24 and PPIA are the most stable genes.
authors.affiliation: Department of Animal Production, Epidemiology and Ecology, University of Turin, Via L. da Vinci 44, 10095 Grugliasco, Italy.
authors.forename: Veronica
authors.initials: V
authors.lastname: Spalenza
authors.forename: Flavia
authors.initials: F
authors.lastname: Girolami
authors.forename: Claudia
authors.initials: C
authors.lastname: Bevilacqua
authors.forename: Fulvio
authors.initials: F
authors.lastname: Riondato
authors.forename: Roberto
authors.initials: R
authors.lastname: Rasero
authors.forename: Carlo
authors.initials: C
authors.lastname: Nebbia
authors.forename: Paola
authors.initials: P
authors.lastname: Sacchi
authors.forename: Patrice
authors.initials: P
authors.lastname: Martin
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism / Q000592:standards; D000465:Algorithms; D000818:Animals; D002417:Cattle / Q000235:genetics*; D021984:Cyclophilin A / Q000235:genetics / Q000378:metabolism / Q000592:standards; D005260:Female; D020869:Gene Expression Profiling / Q000662:veterinary*; D008214:Lymphocytes / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism / Q000592:standards; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards* / Q000662:veterinary; D012015:Reference Standards; D015203:Reproducibility of Results; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism / Q000592:standards
chemical_list: D048948:14-3-3 Proteins; D012333:RNA, Messenger; D012269:Ribosomal Proteins; D021984:Cyclophilin A