Facci Marina R, Auray Gael, Meurens Francois, Buchanan Rachelle, van Kessel Jill, Gerdts Volker
Vaccine & Infectious Disease Organization, University of Saskatchewan, Saskatoon S7N5E3, Canada.
Vet Immunol Immunopathol. 2011 May 15;141(1-2):11-5. doi: 10.1016/j.vetimm.2011.01.005. Epub 2011 Jan 13.
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.
实时定量聚合酶链反应（RT-qPCR）是一种用于评估基因表达变化的关键工具。该工具的准确性依赖于参考基因的选择，这些参考基因在培养条件和刺激后表达保持不变。稳定表达的参考基因用于标准化数据，以便观察到的表达变化不是由于人为因素，而是反映生理变化。在本研究中，我们检测了猪基因甘油醛-3-磷酸脱氢酶（GAPDH）、琥珀酸脱氢酶复合物亚基A（SDHA）、真核延伸因子1γ样蛋白（eEF1）、核糖体蛋白L19（RPL19）、β-肌动蛋白（ACTB）和ATP合酶线粒体F0复合物（ATP5G1）在外周血单核细胞（PBMC）、单核细胞、单核细胞衍生树突状细胞（MoDC）、血液分离树突状细胞（BDC）和T细胞中，在有或无脂多糖（LPS）刺激情况下的表达稳定性。使用geNORM软件确定的M值作为基因稳定性的衡量指标。关于参考基因使用的建议包括：在PBMC中使用GAPDH和β-肌动蛋白；在T细胞中使用RPL19和SDHA；在单核细胞中使用RPL19和β-肌动蛋白；在BDC中使用RPL-19和SDHA；在MoDC中使用RPL-19和ATP5GA。
