Gantasala Nagavara Prasad, Papolu Pradeep Kumar, Thakur Prasoon Kumar, Kamaraju Divya, Sreevathsa Rohini, Rao Uma
Division of Nematology, Indian Agricultural Research Institute, New Delhi 110012, India.
BMC Res Notes. 2013 Aug 6;6:312. doi: 10.1186/1756-0500-6-312.
Analysis of gene expression patterns leads to functional understanding of biological processes. Quantitative real-time PCR has become the most commonly used technique for in-depth studies of gene expression. To quantify variation in specific gene expression, accurate and reliable normalization across different samples and tissues is necessary. This can be achieved by selecting one or more suitable reference genes to compare the target mRNA transcript levels. In the present work, we illustrate the first evaluation of potential internal control or reference genes across different developmental stages of eggplant for reliable quantification of transcripts by real-time PCR.
We have evaluated the stability in expression of six candidate reference genes (18s rRNA, APRT, GAPDH, Cyclophilin, Actin, and RuBP) in a set of tissues representing six developmental stages of eggplant. The candidate genes were cloned from cDNA and analysed by real-time PCR. The expression data analyzed by three statistical methods (geNorm, NormFinder and BestKeeper) identified 18s rRNA, Cyclophilin and APRT as the most stable and suitable reference genes in eggplant. This was further confirmed in four different varieties, two representative lines of transgenic eggplant as well as in nematode infected eggplant.
18s rRNA, Cyclophilin and APRT have been found to be appropriate for the normalization of real-time PCR data for gene expression studies in eggplant.
基因表达模式分析有助于从功能上理解生物学过程。定量实时PCR已成为深入研究基因表达最常用的技术。为了量化特定基因表达的变化，有必要在不同样本和组织之间进行准确可靠的标准化。这可以通过选择一个或多个合适的参考基因来比较目标mRNA转录水平来实现。在本研究中，我们首次评估了茄子不同发育阶段潜在的内参基因或参考基因，以便通过实时PCR可靠地定量转录本。
我们评估了六个候选参考基因（18s rRNA、APRT、GAPDH、亲环蛋白、肌动蛋白和RuBP）在一组代表茄子六个发育阶段的组织中的表达稳定性。从cDNA中克隆候选基因并通过实时PCR进行分析。通过三种统计方法（geNorm、NormFinder和BestKeeper）分析的表达数据确定18s rRNA、亲环蛋白和APRT是茄子中最稳定和合适的参考基因。这在四个不同品种、两个转基因茄子代表性品系以及线虫感染的茄子中得到了进一步证实。
已发现18s rRNA、亲环蛋白和APRT适用于茄子基因表达研究中实时PCR数据的标准化。
