A reliable method for quantification of splice variants using RT-qPCR
A reliable method for quantification of splice variants using RT-qPCR
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DOI：
10.1186/s12867-016-0060-1
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发表时间：
2016-03-15
影响因子：
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通讯作者：
Philipp, Stephan E.
中科院分区：
文献类型：
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作者：
Londono, Julia Camacho;Philipp, Stephan E.
背景：大多数蛋白质异构体来自编码初级RNA转录物的选择性剪接。为了理解单个剪接事件的意义，需要可靠的技术来确定它们的发生率。然而，现有的方法是劳动密集型的，容易出错或有限的use.Results：在这里，我们提出了一种改进的方法来确定在一个单一的网站从选择性剪接产生的转录的相对发病率。使用一步逆转录定量PCR在单个样品内定量剪接变体。将用变体特异性引物对获得的扩增产物与用两种变体共有的引物对获得的扩增产物进行比较。通过熔解曲线分析同时验证变体特异性扩增子的身份。对每种变异的相对发生率进行独立计算。由于变异体的相对发生率必须加起来达到100%，因此该方法提供了一种内部对照，以监测实验误差和均匀的逆转录。该方法的可靠性进行了测试，使用混合物的cDNA模板以及RNA样品从不同的sources.Conclusion：这里描述的方法，是很容易建立，不需要无关的参考基因和耗时，容易出错的标准曲线。它提供了一种可靠和精确的技术来区分两种剪接变异体相对发生率的微小差异。
Background: The majority of protein isoforms arise from alternative splicing of the encoding primary RNA transcripts. To understand the significance of single splicing events, reliable techniques are needed to determine their incidence. However, existing methods are labour-intensive, error-prone or of limited use.Results: Here, we present an improved method to determine the relative incidence of transcripts that arise from alternative splicing at a single site. Splice variants were quantified within a single sample using one-step reverse transcription quantitative PCR. Amplification products obtained with variant specific primer pairs were compared to those obtained with primer pairs common to both variants. The identities of variant specific amplicons were simultaneously verified by melt curve analysis. Independent calculations of the relative incidence of each variant were performed. Since the relative incidences of variants have to add upto 100 %, the method provides an internal control to monitor experimental errors and uniform reverse transcription. The reliability of the method was tested using mixtures of cDNA templates as well as RNA samples from different sources.Conclusion: The method described here, is easy to set up and does not need unrelated reference genes and time consuming, error-prone standard curves. It provides a reliable and precise technique to distinguish small differences of the relative incidence of two splice variants.