Validation of endogenous reference genes in Buglossoides arvensis for normalizing RT-qPCR-based gene expression data.
Validation of endogenous reference genes in Buglossoides arvensis for normalizing RT-qPCR-based gene expression data.
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DOI：
10.1186/s40064-015-0952-4
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Filion M
中科院分区：
文献类型：
--
作者：
Gadkar VJ;Filion M
稳定表达的参考基因（RG）的选择是产生可靠且可重复的定量实时逆转录聚合酶链反应（RT-qPCR）基因表达数据的重要步骤。在这项研究中，我们试图验证Buglossoides arvensis的RG，这是一种高营养价值的植物，其精炼籽油以商业商品名Ahiflower™进入市场。这种杂草植物因其在其种子中显著积累多不饱和脂肪酸（PUFA）十八碳四烯酸（SDA，C18：4 n-3）的天然能力而受到关注，这对于大多数植物物种来说是不常见的。从B中分离得到10个候选RGs（β-Act、18 S rRNA、EF-1 a、α-TuB b、UBQ、α-actin、CAC、PP 2a、RUBISCO、GAPDH）。设计了符合农杆菌和TaqMan™的引物/探针用于RT-qPCR分析。在不同的组织和生长状态下分析这些基因转录本的缺失。两个最广泛使用的算法，geNorm和Normalization，显示这些RG的表达水平的变化。然而，对结果的组合分析清楚地将CAC和α-肌动蛋白分别确定为最稳定和最不稳定的RG候选物。本研究首次在非模型系统B中识别和验证了RG。arvensis，一种杂草植物，预计将成为膳食omega-3 PUFA的重要但可持续的来源。本文的在线版本（doi：10.1186/s40064-015-0952-4）包含补充材料，可供授权用户使用。
Selection of a stably expressed reference gene (RG) is an important step for generating reliable and reproducible quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) gene expression data. We, in this study, have sought to validate RGs for Buglossoides arvensis, a high nutraceutical value plant whose refined seed oil is entering the market under the commercial trade name Ahiflower™. This weed plant has received attention for its natural ability to significantly accumulate the poly-unsaturated fatty acid (PUFA) stearidonic acid (SDA, C18:4n-3) in its seeds, which is uncommon for most plant species. Ten candidate RGs (β-Act, 18S rRNA, EF-1a, α-Tub, UBQ, α-actin, CAC, PP2a, RUBISCO, GAPDH) were isolated from B. arvensis and TaqMan™ compliant primers/probes were designed for RT-qPCR analysis. Abundance of these gene transcripts was analyzed across different tissues and growth regimes. Two of the most widely used algorithms, geNorm and NormFinder, showed variation in expression levels of these RGs. However, combinatorial analysis of the results clearly identified CAC and α-actin as the most stable and unstable RG candidates, respectively. This study has for the first time identified and validated RGs in the non-model system B. arvensis, a weed plant projected to become an important yet sustainable source of dietary omega-3 PUFA. The online version of this article (doi:10.1186/s40064-015-0952-4) contains supplementary material, which is available to authorized users.
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