Accurate quantitative RT-PCR for relative expression of Slo splice variants.
Accurate quantitative RT-PCR for relative expression of Slo splice variants.
复制标题
用于 Slo 剪接变体相对表达的准确定量 RT-PCR。
DOI：
10.1016/s0165-0270(02)00015-8
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发表时间：
2002
影响因子：
3
通讯作者：
McCobb,DavidP
中科院分区：
文献类型：
--
作者：
Mahmoud,SaharF;Bezzerides,AlexL;Riba,Rebecca;Lai,Guey-Jen;Lovell,PeterV;Hara,Yuko;McCobb,DavidP
人们对使用多模板逆转录聚合酶链反应（RT-PCR）作为低丰度mrna的定量工具非常感兴趣。为了实现离子通道基因中一个剪接决定的应激和激素相关调控的精细分级量化，我们测试了同时扩增两个剪接变异体的可靠性，同时扩增一对侧面组成引物。出乎意料的是，两种扩增产物之间不分皂白的异双工，尽管有很大的长度差异，以及它们与一个同双工的紧密结合，要求严格的变性电泳方案。方便的是，一种对单链DNA具有高亲和力的新型荧光染料已经出现。虽然染料具有良好的动态范围，但我们发现染料和凝胶的饱和度由于产物之间的长度差异而导致相对丰度的计算存在不对称误差。避免了几个陷阱，染料校准可以用来纠正错误。我们还发现，两种模板的扩增效率差异不是恒定的，而是依赖于初始模板比例，需要非线性校正。总之，这些改进为我们提供了非常一致的定量结果，从而推进了我们对激素机制的分析，这些机制是调控离子通道选择性剪接的基础，而离子通道的选择性剪接与应激反应密切相关。
Much interest has been shown in the use of multi-template reverse transcription-polymerase chain reaction (RT-PCR) as a quantitative instrument for low-abundance mRNAs. A desire to achieve finely-graded quantification of the stress- and hormone-related regulation of one splicing decision in an ion channel gene motivated us to test the reliability of simultaneous amplification of two splice variants with one pair of flanking constitutive primers. Unexpectedly indiscriminate heteroduplexing between the two amplification products, despite a large length difference, and their tight comigration with one homoduplex, mandated a rigorously-denaturing electrophoresis protocol. Conveniently, a new fluorescent dye with high affinity for single-stranded DNA has become available. Though the dye has a good dynamic range, we found that dye and gel saturation compounded by the length difference between products introduced an asymmetrical error into the calculation of relative abundance. Avoiding several pitfalls, dye calibration could be used to correct the error. We also found that differences in the amplification efficiency of the two templates were not constant, but dependent on the initial template ratio, requiring a non-linear correction. Together these improvements gave us very consistent quantitative results, and thus advance our analysis of hormonal mechanisms underlying the regulation of alternative splicing of an ion channel critically involved in stress responses.