Ct shift: A novel and accurate real-time PCR quantification model for direct comparison of different nucleic acid sequences and its application for transposon quantifications
Ct shift: A novel and accurate real-time PCR quantification model for direct comparison of different nucleic acid sequences and its application for transposon quantifications
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DOI：
10.1016/j.gene.2016.10.035
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发表时间：
2017-01-20
期刊：
影响因子：
3.5
通讯作者：
Orban, Tamas I.
中科院分区：
文献类型：
--
作者：
Kolacsek, Orsolya;Pergel, Eniko;Orban, Tamas I.
定量PCR在诊断和基础研究中有许多应用。与许多其他技术一样，定量的基础是在同一实体的不同（未知和已知或参考）标本之间进行比较。当目的是比较样品中不同物种的真实的数量时，人们无法逃避它们各自的精确绝对定量。我们已经建立了一个简单可靠的方法（Ct移位法），它结合了绝对和相对的方法。它需要含有待比较的两种扩增子序列的质粒标准品（例如，目标靶标和内源性对照品）。它可以作为两个靶标的模板拷贝数相等的参考样品。使用Delta Delta Ct公式，我们可以量化每个未知样品中两种模板的确切比例。Ct位移法已成功应用于转座子基因拷贝测量，以及用于比较cDNA样品中的不同mRNA。本研究提供了概念证明，并介绍了该方法的一些潜在应用;即使不需要真实的参比样品，结果的绝对性也有助于该方法的通用性和不同研究的可比性。(C)2016爱思唯尔B. V.保留所有权利。
There are numerous applications of quantitative PCR for both diagnostic and basic research. As in many other techniques the basis of quantification is that comparisons are made between different (unknown and known or reference) specimens of the same entity. When the aim is to compare real quantities of different species in samples, one cannot escape their separate precise absolute quantification. We have established a simple and reliable method for this purpose (Ct shift method) which combines the absolute and the relative approach. It requires a plasmid standard containing both sequences of amplicons to be compared (e.g. the target of interest and the endogenous control). It can serve as a reference sample with equal copies of templates for both targets. Using the Delta Delta Ct formula we can quantify the exact ratio of the two templates in each unknown sample. The Ct shift method has been successfully applied for transposon gene copy measurements, as well as for comparison of different mRNAs in cDNA samples. This study provides the proof of concept and introduces some potential applications of the method; the absolute nature of results even without the need for real reference samples can contribute to the universality of the method and comparability of different studies. (C) 2016 Elsevier B.V. All rights reserved.