High-resolution semi-quantitative real-time PCR without the use of a standard curve
High-resolution semi-quantitative real-time PCR without the use of a standard curve
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DOI：
10.2144/01313st03
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发表时间：
2001-09-01
期刊：
影响因子：
2.7
通讯作者：
McBrien, NA
中科院分区：
文献类型：
--
作者：
Gentle, A;Anastasopoulos, F;McBrien, NA
研究了一种简单、适应性强、半定量、实时RT-PCR检测方法的重复性和灵敏度。只要扩增条件是特定于所要扩增的PCR产物，该分析可以轻松快速地用于定量任何基因产物的相对水平，而无需使用标准品。利用Light-Cycler （TM）实时PCR机，扩增了379 bp cDNA模板的连续10倍稀释序列（跨越4个数量级），并使用SYBR (R) Green I化学对PCR产物进行缺陷检测。第二天又重复了这个实验。实验设计是这样的，数据可以用适当的方法进行分析，以测试可重复性。研究发现，对于三次重复的样品，在一次分析中，可以可靠地检测到23%的差异。此外，当考虑到导致分析变异性的所有因素时，例如移液和扩增效率的日常变化，使用4个样本量可以检测到52%的目标模板差异。结果发现，该测定法至少在四个数量级上呈线性。
The repeatability and sensitivity of a simple, adaptable, semi-quantitative, realtime RT-PCR assay was investigated. The assay can be easily and rapidly applied to quantitate relative levels of any gene product without using standards, provided that amplification conditions are specific for the PCR product of interest. Using the Light-Cycler (TM) real-time PCR machine, a serial 10-fold dilution series, (spanning four orders of magnitude) of a 379-bp cDNA template was amplified, and the PCR product was defected using SYBR (R) Green I chemistry. The experiment was repeated on a subsequent day. The experimental design was such that the data lent itself to analysis using an appropriate method for testing repeatability. It was found that, within a single assay for samples assayed in triplicate, a difference of 23% may be reliably detected Furthermore, when all of the factors that contribute to variability in the assay are taken into account, such as day-to-day variation in pipetting and amplification efficiency, a 52% difference in target template can be detected using a sample size of 4. The assay was found to be linear over at least four orders of magnitude.