Selection and validation of reference genes for quantitative gene expression studies by real-time PCR in eggplant (Solanum melongena L).
Selection and validation of reference genes for quantitative gene expression studies by real-time PCR in eggplant (Solanum melongena L).
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DOI：
10.1186/1756-0500-6-312
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发表时间：
2013-08-06
影响因子：
1.8
通讯作者：
Rao U
中科院分区：
文献类型：
--
作者：
Gantasala NP;Papolu PK;Thakur PK;Kamaraju D;Sreevathsa R;Rao U
基因表达模式的分析导致对生物过程的功能性理解。实时定量PCR已成为深入研究基因表达的最常用技术。为了量化特定基因表达的变异，需要对不同样本和组织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靶mRNA转录物水平来实现。在目前的工作中，我们说明了第一次评估潜在的内部控制或参考基因在茄子的不同发育阶段，通过实时PCR可靠的量化成绩单。我们已经评估了6个候选参考基因（18S rRNA，APRT，GAPDH，亲环素，肌动蛋白，RuBP）在一组代表茄子6个发育阶段的组织中表达的稳定性。从cDNA中克隆候选基因，并通过实时PCR进行分析。通过geNorm、Normands和BestKeeper 3种统计方法对表达数据进行分析，确定18SrRNA、Cyclophilin和APRT为茄子中最稳定、最合适的参考基因。这在四个不同品种、两个代表性的转基因茄子品系以及线虫感染的茄子中得到进一步证实。已发现18s rRNA、亲环素和APRT适用于茄子基因表达研究的实时PCR数据的标准化。
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.