Anstaett Olivia L, Brownlie Joe, Collins Margaret E, Thomas Carole J
Department of Pathology and Infectious Disease, The Royal Veterinary College, University of London, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire AL9 7TA, UK.
Vet Immunol Immunopathol. 2010 Oct 15;137(3-4):201-7. doi: 10.1016/j.vetimm.2010.05.006. Epub 2010 Jun 1.
Reverse transcription quantitative PCR (RT-qPCR) is a highly sensitive tool that can be used for accurate and reliable gene expression analysis; however, careful normalisation to a set of stably expressed endogenous reference genes is essential. Expression levels of many reference genes in RT-qPCR analyses can be extremely variable under different experimental conditions, producing potentially erroneous results (Bustin, 2002). This limitation can be overcome with a systematic evaluation of candidate reference genes to determine the most stable. In the present study eight candidate reference genes were evaluated in a bovine lymphoid (BL-3) cell culture system over seven different time points in response to three different Bovine Viral Diarrhoea Virus (BVDV) strains. Data were analysed using BestKeeper (Pfaffl et al., 2004), geNorm (Vandesompele et al., 2002), and NormFinder (Andersen et al., 2004) validation programs and results enable the candidate reference genes to be ranked from most to least stable. Quantification cycle (C(q)) variability was determined between samples, i.e. between treatment groups and time points, and variability was also observed between the three validation programs. The reference gene combination of beta-actin and hypoxanthine-guanine phosphoribosyl transferase (HPRT) was found to be the most stable in Norm Finder. BestKeeper and geNorm both demonstrated beta-microglobulin and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase (YWHAZ) as the most stable. The determination of a stable set of reference genes in the BL-3 cell culture system facilitates analysis of expression levels for appropriate genes of interest. This study further emphasises the need to accurately validate candidate reference genes before use in gene expression RT-qPCR studies.
逆转录定量聚合酶链反应（RT-qPCR）是一种高度灵敏的工具，可用于准确可靠的基因表达分析；然而，仔细地将其标准化为一组稳定表达的内源性参照基因至关重要。在RT-qPCR分析中，许多参照基因的表达水平在不同实验条件下可能极具变异性，从而产生潜在的错误结果（Bustin，2002年）。通过对候选参照基因进行系统评估以确定最稳定的基因，可克服这一局限性。在本研究中，在牛淋巴细胞（BL-3）细胞培养系统中，针对七个不同时间点，对三种不同的牛病毒性腹泻病毒（BVDV）毒株，评估了八个候选参照基因。使用BestKeeper（Pfaffl等人，2004年）、geNorm（Vandesompele等人，2002年）和NormFinder（Andersen等人，2004年）验证程序对数据进行分析，结果能够将候选参照基因从最稳定到最不稳定进行排序。确定了样本之间（即处理组和时间点之间）的定量循环（C(q)）变异性，并且在三个验证程序之间也观察到了变异性。在Norm Finder中，发现β-肌动蛋白和次黄嘌呤-鸟嘌呤磷酸核糖基转移酶（HPRT）的参照基因组合最稳定。BestKeeper和geNorm均表明β-微球蛋白和酪氨酸3-单加氧酶/色氨酸5-单加氧酶（YWHAZ）最稳定。在BL-3细胞培养系统中确定一组稳定的参照基因有助于分析感兴趣的适当基因的表达水平。本研究进一步强调了在基因表达RT-qPCR研究中使用前准确验证候选参照基因的必要性。
