title: Stability of expression of reference genes in porcine peripheral blood mononuclear and dendritic cells.
abstract: Real-time quantitative PCR (RT-qPCR) is a critical tool used to evaluate changes in gene expression. The precision of this tool is reliant upon the selection of reference genes whose expression remains unaltered in culture conditions and following stimulation. Stably expressed reference genes are used to normalize data so observed changes in expression are not due to artifacts but rather reflect physiological changes. In this study, we examined the expression stability of the porcine genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex subunit A (SDHA), eukaryotic elongation factor 1 gamma-like protein (eEF1), ribosomal protein L19 (RPL19), beta-actin (ACTB) and ATP synthase mitochondrial F0 complex (ATP5G1) in peripheral blood mononuclear cells (PBMCs), monocytes, monocyte-derived dendritic cells (MoDCs), blood isolated dendritic cells (BDCs) and T cells with or without stimulation with lipolysaccharide (LPS). An M value was used as a measure of gene stability as determined using geNORM software. Recommendations for the use of reference genes include using GAPDH and B-actin in PBMCs: RPL19 and SDHA in T cells; RPL19 and B-actin in monocytes; RPL-19 and SDHA in BDCs: and RPL-19 and ATP5GA in MoDCs.
authors.affiliation: Vaccine & Infectious Disease Organization, University of Saskatchewan, Saskatoon S7N5E3, Canada.
authors.forename: Marina R
authors.initials: MR
authors.lastname: Facci
authors.forename: Gael
authors.initials: G
authors.lastname: Auray
authors.forename: Francois
authors.initials: F
authors.lastname: Meurens
authors.forename: Rachelle
authors.initials: R
authors.lastname: Buchanan
authors.forename: Jill
authors.initials: J
authors.lastname: van Kessel
authors.forename: Volker
authors.initials: V
authors.lastname: Gerdts
mesh_terms: D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D003713:Dendritic Cells / Q000276:immunology*; D015977:Eukaryotic Initiation Factor-1 / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation / Q000276:immunology*; D005796:Genes / Q000235:genetics; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000096:biosynthesis / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D025261:Mitochondrial Proton-Translocating ATPases / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012269:Ribosomal Proteins / Q000096:biosynthesis / Q000235:genetics; D013385:Succinate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D013552:Swine / Q000235:genetics / Q000276:immunology
chemical_list: D000199:Actins; D015977:Eukaryotic Initiation Factor-1; D012333:RNA, Messenger; D012269:Ribosomal Proteins; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+); D013385:Succinate Dehydrogenase; D025261:Mitochondrial Proton-Translocating ATPases