Selection of reference genes for transcriptional analysis of edible tubers of potato (Solanum tuberosum L.).
Selection of reference genes for transcriptional analysis of edible tubers of potato (Solanum tuberosum L.).
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DOI：
10.1371/journal.pone.0120854
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Frazzon J
中科院分区：
文献类型：
--
作者：
Mariot RF;de Oliveira LA;Voorhuijzen MM;Staats M;Hutten RC;Van Dijk JP;Kok E;Frazzon J
马铃薯(Solanum Tuberosum)产量在过去50年里大幅增加，这是通过改进农艺和生物技术努力相结合实现的。基因研究正在进行，以提高新的品质和开发新的品种。逆转录酶定量聚合酶链式反应(RT-qPCR)是一种用于基因表达分析的标杆分析工具，但其准确性高度依赖于不变参考基因的可靠归一化策略。为此，本工作的目的是筛选和验证马铃薯块茎转录分析的参考基因。为此，根据RNA-Seq实验结果，针对马铃薯块茎中相对稳定表达的10个基因设计了RT-qPCR引物。设计了跨越外显子边界的引物，以避免基因组DNA污染。在geNorm、NormFinder和BestKeeper算法确定的候选基因的排名中观察到了差异。GeNorm和NormFinder测定的序列非常相似，对于所有样品来说，最稳定的候选者是C2、胞囊复合体组分Sec3(SEC3)和ATCUL3/ATCUL3A/CUL3/CUL3A(CUL3A)。根据BestKeeper的说法，进口素α和泛素相关/ts-n基因最稳定。在RT-qPCR研究中，选择了3个基因作为马铃薯食用块茎的参考基因。第一个称为C2，是由NormFinder和geNorm共同选择的，第二个是SEC3，由NormFinder选择，第三个是CUL3A，由geNorm选择。这项工作中确定的适当参考基因将有助于提高基因表达定量分析的准确性，因为它考虑了样本之间可能观察到的RNA质量或反转录效率的差异。
Potato (Solanum tuberosum) yield has increased dramatically over the last 50 years and this has been achieved by a combination of improved agronomy and biotechnology efforts. Gene studies are taking place to improve new qualities and develop new cultivars. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a bench-marking analytical tool for gene expression analysis, but its accuracy is highly dependent on a reliable normalization strategy of an invariant reference genes. For this reason, the goal of this work was to select and validate reference genes for transcriptional analysis of edible tubers of potato. To do so, RT-qPCR primers were designed for ten genes with relatively stable expression in potato tubers as observed in RNA-Seq experiments. Primers were designed across exon boundaries to avoid genomic DNA contamination. Differences were observed in the ranking of candidate genes identified by geNorm, NormFinder and BestKeeper algorithms. The ranks determined by geNorm and NormFinder were very similar and for all samples the most stable candidates were C2, exocyst complex component sec3 (SEC3) and ATCUL3/ATCUL3A/CUL3/CUL3A (CUL3A). According to BestKeeper, the importin alpha and ubiquitin-associated/ts-n genes were the most stable. Three genes were selected as reference genes for potato edible tubers in RT-qPCR studies. The first one, called C2, was selected in common by NormFinder and geNorm, the second one is SEC3, selected by NormFinder, and the third one is CUL3A, selected by geNorm. Appropriate reference genes identified in this work will help to improve the accuracy of gene expression quantification analyses by taking into account differences that may be observed in RNA quality or reverse transcription efficiency across the samples.
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