De Boever S, Vangestel C, De Backer P, Croubels S, Sys S U
Department of Pharmacology, Toxicology, Biochemistry and Organ Physiology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium.
Vet Immunol Immunopathol. 2008 Apr 15;122(3-4):312-7. doi: 10.1016/j.vetimm.2007.12.002. Epub 2008 Jan 3.
Real-time PCR has become a powerful tool for the detection of inflammatory parameters, including cytokines. Reference or housekeeping genes are used for the normalization of real-time RT-PCR results. In order to obtain reliable results, the stability of these housekeeping genes needs to be determined. In this study the stability of five genes, including beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phophoribosyl-transferase (HPRT), ubiquitin (UB) and glucose-6-phosphate dehydrogenase (G6PDH), was determined in a lipopolysaccharide inflammation model in chickens. beta-Actin appeared to be the most stable single gene in our model. Because the use of a single gene for normalization can lead to relatively large errors, the use of the geometric mean of multiple reference genes or normalization factor is preferred. The most stable combination for gene expression analysis in this lipopolysaccharide inflammation model in chickens is G6PDH and UB, since their correlation coefficients were 0.953 and 0.969, respectively (BestKeeper) and an M value of 0.34 and a low V(2/3) value of 0.155 (geNorm) were obtained. The use of HPRT and GAPDH should be avoided. The stable housekeeping genes, G6PDH and UB together, can be used to normalize the expression of pro-inflammatory cytokines in a lipopolysaccharide inflammation model in chickens.
实时定量聚合酶链反应（Real-time PCR）已成为检测包括细胞因子在内的炎症参数的有力工具。参照基因或管家基因用于实时逆转录聚合酶链反应（RT-PCR）结果的标准化。为了获得可靠的结果，需要确定这些管家基因的稳定性。在本研究中，在鸡的脂多糖炎症模型中测定了包括β-肌动蛋白、甘油醛-3-磷酸脱氢酶（GAPDH）、次黄嘌呤磷酸核糖转移酶（HPRT）、泛素（UB）和葡萄糖-6-磷酸脱氢酶（G6PDH）在内的五个基因的稳定性。在我们的模型中，β-肌动蛋白似乎是最稳定的单个基因。由于使用单个基因进行标准化可能会导致相对较大的误差，因此首选使用多个参照基因的几何平均值或标准化因子。在鸡的这种脂多糖炎症模型中，用于基因表达分析的最稳定组合是G6PDH和UB，因为它们的相关系数分别为0.953和0.969（BestKeeper），并且获得的M值为0.34，低V(2/3)值为0.155（geNorm）。应避免使用HPRT和GAPDH。稳定的管家基因G6PDH和UB一起可用于标准化鸡脂多糖炎症模型中促炎细胞因子的表达。
