Statistical tools for transgene copy number estimation based on real-time PCR
Statistical tools for transgene copy number estimation based on real-time PCR
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DOI：
10.1186/1471-2105-8-s7-s6
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发表时间：
2007-01-01
影响因子：
3
通讯作者：
Stewart, C. Neal, Jr.
中科院分区：
文献类型：
--
作者：
Yuan, Joshua S.;Burris, Jason;Stewart, C. Neal, Jr.
背景资料：与传统的转基因拷贝数检测技术如Southern印迹分析相比，实时PCR提供了一种快速、廉价和高通量的替代方法。然而，基于实时PCR的转基因拷贝数估计往往是模糊的和主观的，这是由于缺乏适当的统计分析和数据质量控制来提供具有预测值的拷贝数的可靠估计。尽管最近的进展，在统计分析的实时PCR，很少有出版物已经集成了这些进展，在实时PCR为基础的转基因拷贝数determination.Results：三个实验设计和四个数据质量控制集成的统计模型。对于第一种方法，基于连续稀释的模板建立转基因的外部校准曲线。比较来自对照转基因事件和推定的转基因事件的Ct数以得出转基因拷贝数或接合性估计。简单的线性回归和两组T检验程序相结合，从这个设计的数据建模。对于第二个实验设计，生成内部参考基因和转基因的标准曲线，并将转基因的拷贝数与内部参考基因的拷贝数进行比较。多元回归模型和方差分析模型可以用来分析数据和执行质量控制的这种方法。在第三个实验设计中，转基因拷贝数与参考基因进行比较，没有标准曲线，而是直接基于荧光数据。基于两种不同的扩增效率积分方法，提出了两种不同的多元回归模型来分析数据。我们的研究结果突出了适当的统计处理和质量控制集成在实时PCR为基础的转基因拷贝数determination.Conclusion的重要性：这些统计方法允许实时PCR为基础的转基因拷贝数估计是更可靠和精确的一个适当的统计估计。正确的置信区间是明确预测转基因拷贝数所必需的。比较了四种不同的统计方法的优缺点。此外，统计学方法也可以应用于其他基于实时PCR的定量分析，包括转染效率分析和病原体定量。
Background: As compared with traditional transgene copy number detection technologies such as Southern blot analysis, real-time PCR provides a fast, inexpensive and high-throughput alternative. However, the real-time PCR based transgene copy number estimation tends to be ambiguous and subjective stemming from the lack of proper statistical analysis and data quality control to render a reliable estimation of copy number with a prediction value. Despite the recent progresses in statistical analysis of real-time PCR, few publications have integrated these advancements in real-time PCR based transgene copy number determination.Results: Three experimental designs and four data quality control integrated statistical models are presented. For the first method, external calibration curves are established for the transgene based on serially-diluted templates. The Ct number from a control transgenic event and putative transgenic event are compared to derive the transgene copy number or zygosity estimation. Simple linear regression and two group T-test procedures were combined to model the data from this design. For the second experimental design, standard curves were generated for both an internal reference gene and the transgene, and the copy number of transgene was compared with that of internal reference gene. Multiple regression models and ANOVA models can be employed to analyze the data and perform quality control for this approach. In the third experimental design, transgene copy number is compared with reference gene without a standard curve, but rather, is based directly on fluorescence data. Two different multiple regression models were proposed to analyze the data based on two different approaches of amplification efficiency integration. Our results highlight the importance of proper statistical treatment and quality control integration in real-time PCR-based transgene copy number determination.Conclusion: These statistical methods allow the real-time PCR-based transgene copy number estimation to be more reliable and precise with a proper statistical estimation. Proper confidence intervals are necessary for unambiguous prediction of trangene copy number. The four different statistical methods are compared for their advantages and disadvantages. Moreover, the statistical methods can also be applied for other real-time PCR-based quantification assays including transfection efficiency analysis and pathogen quantification.