Ish-Shalom Shahar, Lichter Amnon
Department of Post Harvest Science, ARO, The Volcani Center, Bet Dagan, Israel.
Methods Mol Biol. 2010;638:103-14. doi: 10.1007/978-1-60761-611-5_7.
The Real-Time quantitative PCR (qPCR) method has become central for the quantification of gene expression as well as other applications. The major advantages of qPCR are the utilization of small amount of template, high sensitivity and the ability to detect products during the reaction. After selecting qPCR among other options (northern blot, semi-quantitative PCR), one should consider several factors. The first and critical step in qPCR of fungi is the selection of an appropriate growth medium and RNA extraction method, which will avoid accumulation of inhibitors. In this chapter, we focus on detection of the accumulating product with the Syber Green dye, but other detection technologies, such as hybridization probes, might be considered as well. Accurate qPCR analysis with Syber Green depends mainly on optimal PCR reaction, and therefore it is important to design primers that will avoid formation of interfering structures. It is possible to use absolute quantification of the template in the sample, or to conduct a relative analysis, as described in this protocol. In the relative analysis method, expression of the gene of interest is compared with expression of a reference gene. According to our experience as well as according to the literature, it is recommended to use at least three reference genes in order to obtain reliable results.
实时定量PCR（qPCR）方法已成为基因表达定量及其他应用的核心方法。qPCR的主要优点是使用少量模板、灵敏度高以及能够在反应过程中检测产物。在从其他方法（Northern印迹、半定量PCR）中选择qPCR后，应考虑几个因素。真菌qPCR的第一步也是关键步骤是选择合适的生长培养基和RNA提取方法，这将避免抑制剂的积累。在本章中，我们重点介绍使用Syber Green染料检测积累的产物，但也可以考虑其他检测技术，如杂交探针。使用Syber Green进行准确的qPCR分析主要取决于优化的PCR反应，因此设计避免形成干扰结构的引物很重要。可以对样品中的模板进行绝对定量，也可以按照本方案进行相对分析。在相对分析方法中，将感兴趣基因的表达与参考基因的表达进行比较。根据我们的经验以及文献，建议使用至少三个参考基因以获得可靠的结果。
