Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.
Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.
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DOI：
10.1371/journal.pone.0075850
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Yang L
中科院分区：
文献类型：
--
作者：
Huang H;Cheng F;Wang R;Zhang D;Yang L
正确选择内源参考基因及其实时 PCR 检测对于转基因生物 (GMO) 检测非常重要。为了寻找适合普通小麦（Triticum aestivum L.）DNA含量或拷贝数定量的内源内参基因及其实时PCR检测方法，对之前报道的4个小麦内源内参基因及其实时PCR检测方法进行了综合评估，以了解37个普通小麦品系中的目标基因序列变异及其实时PCR性能。在PKABA1和ALMT1基因中观察到3个SNP，这些SNP显着降低了实时PCR扩增的效率。 GeNorm 对常见小麦品系中各基因的实时 PCR 性能分析表明，Waxy-D1 检测在所有测试品系中具有最低的 M 值和最好的稳定性。所有结果表明Waxy-D1基因及其实时PCR检测最适合用作普通小麦DNA含量定量的内源内参基因。经过验证的 Waxy-D1 基因检测将有助于建立准确且可靠的转基因小麦定性和定量 PCR 分析。
Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.
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