Expression profiling by real-time quantitative polymerase chain reaction (RT-qPCR).
Expression profiling by real-time quantitative polymerase chain reaction (RT-qPCR).
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DOI：
10.1007/978-1-4939-0882-0_13
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发表时间：
2014-01-01
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影响因子：
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通讯作者：
Anders, Hans-Joachim
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文献类型：
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作者：
Lech, Maciej;Anders, Hans-Joachim
实时定量PCR是通常用于定量核酸的标准PCR技术的变体。然而，在该技术中，扩增的特定序列的量可以在PCR循环的每个阶段定量。如果特定样品中存在大量拷贝的研究序列，则在早期循环中已检测到扩增产物;如果序列罕见，则在后期循环中观察到扩增。使用荧光探针或荧光DNA结合染料获得扩增产物的定量。荧光信号的积累可以在PCR反应的35-45个循环中的每个循环期间通过实时PCR仪器测量，这通过消除使用凝胶电泳的扩增产物的可视化而简化了程序。实时PCR允许在PCR反应期间一旦可检测到产物就对已经存在的产物的量进行定量。如果正确执行，该方法可用于生命科学、医学和诊断学中的精确基因表达分析，并已成为mRNA定量的标准选择方法。然而，在过去的几年中，很明显，实时PCR是复杂的，RNA模板的可变性，测定设计，不适当的数据标准化和数据解释可能会导致各种分析问题。
Real-time quantitative PCR is a variation of the standard PCR technique that is commonly used to quantify nucleic acid. However, in this technique the amount of amplified specific sequence can be quantified at each stage of the PCR cycle. If investigated sequence is present in large number of copies in particular sample, amplification product is detected already in earlier cycles; if the sequence is rare, amplification is observed in later cycles. Quantification of amplified product is acquired using fluorescent probes or fluorescent DNA-binding dyes. Accumulation of fluorescent signal can be measured by real-time PCR instruments during each of 35-45 cycwwles of the PCR reaction, which simplify the procedure by eliminating the visualization of the amplified products using gel electrophoresis. Real-time-PCR allows quantifying the amount of product already during the PCR reaction as soon as it is detectable. Correctly performed, this method may be used for precise gene expression analysis in life science, medicine, and diagnostics and has become the standard method of choice for the quantification of mRNA. However in the past few years it became obvious that real-time PCR is complex and variability of RNA templates, assay designs, inappropriate data normalization, and data interpretation may cause diverse analytical problems.