National Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (East China), Ministry of Agriculture of PR China, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, PR China.
Biochem Biophys Res Commun. 2012 Aug 3;424(3):398-403. doi: 10.1016/j.bbrc.2012.06.119. Epub 2012 Jul 3.
Real-time quantitative reverse transcription PCR (RT-qPCR) is a rapid and reliable method for gene expression studies. Normalization based on reference genes can increase the reliability of this technique; however, recent studies have shown that almost no single reference gene is universal for all possible experimental conditions. In this study, eight frequently used reference genes were investigated, including Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Actin2/7 (ACT), Tubulin alpha-5 (TUA), Tubulin beta-1 (TUB), 18S ribosomal RNA (18SrRNA), RNA polymerase-II transcription factor (RPII), Elongation factor 1-b (EF-1b) and Translation elongation factor 2 (TEF2). Expression stability of candidate reference genes was examined across 27 radish samples, representing a range of tissue types, cultivars, photoperiodic and vernalization treatments, and developmental stages. The eight genes in these sample pools displayed a wide range of Ct values and were variably expressed. Two statistical software packages, geNorm and NormFinder showed that TEF2, RPII and ACT appeared to be relatively stable and therefore the most suitable for use as reference genes. These results facilitate selection of desirable reference genes for accurate gene expression studies in radish.
实时荧光定量逆转录聚合酶链反应 (RT-qPCR) 是一种快速可靠的基因表达研究方法。基于参考基因的标准化可以提高该技术的可靠性；然而，最近的研究表明，几乎没有单一的参考基因适用于所有可能的实验条件。在这项研究中，研究了包括甘油醛-3-磷酸脱氢酶 (GAPDH)、肌动蛋白 2/7 (ACT)、微管蛋白 alpha-5 (TUA)、微管蛋白 beta-1 (TUB)、18S 核糖体 RNA (18SrRNA)、RNA 聚合酶-II 转录因子 (RPII)、延伸因子 1-b (EF-1b) 和翻译延伸因子 2 (TEF2) 在内的 8 个常用参考基因。在 27 个萝卜样本中，研究了候选参考基因的表达稳定性，这些样本代表了一系列组织类型、品种、光周期和春化处理以及发育阶段。在这些样本池中，这 8 个基因的 Ct 值差异很大，表达情况也不同。两个统计软件包 geNorm 和 NormFinder 显示，TEF2、RPII 和 ACT 似乎相对稳定，因此最适合用作参考基因。这些结果有助于选择合适的参考基因，以在萝卜中进行准确的基因表达研究。
