Ahmed Mahmoud, Kim Deok Ryong
Department of Biochemistry and Convergence Medical Sciences and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju, Gyeongnam, South Korea.
PeerJ. 2018 Mar 16;6:e4473. doi: 10.7717/peerj.4473. eCollection 2018.
Real-time quantitative PCR (qPCR) is a broadly used technique in the biomedical research. Currently, few different analysis models are used to determine the quality of data and to quantify the mRNA level across the experimental conditions.
We developed an R package to implement methods for quality assessment, analysis and testing qPCR data for statistical significance. Double Delta and standard curve models were implemented to quantify the relative expression of target genes from in standard qPCR control-group experiments. In addition, calculation of amplification efficiency and curves from serial dilution qPCR experiments are used to assess the quality of the data. Finally, two-group testing and linear models were used to test for significance of the difference in expression control groups and conditions of interest.
Using two datasets from qPCR experiments, we applied different quality assessment, analysis and statistical testing in the pcr package and compared the results to the original published articles. The final relative expression values from the different models, as well as the intermediary outputs, were checked against the expected results in the original papers and were found to be accurate and reliable.
The pcr package provides an intuitive and unified interface for its main functions to allow biologist to perform all necessary steps of qPCR analysis and produce graphs in a uniform way.
实时定量聚合酶链反应（qPCR）是生物医学研究中广泛使用的技术。目前，很少有不同的分析模型用于确定数据质量并在不同实验条件下对mRNA水平进行定量。
我们开发了一个R包，以实现用于qPCR数据质量评估、分析和统计显著性检验的方法。在标准qPCR对照组实验中，采用双delta法和标准曲线模型对靶基因的相对表达进行定量。此外，通过对系列稀释qPCR实验的扩增效率和曲线进行计算来评估数据质量。最后，使用两组检验和线性模型来检验感兴趣的表达对照组和条件之间差异的显著性。
使用来自qPCR实验的两个数据集，我们在pcr包中应用了不同的质量评估、分析和统计检验，并将结果与原始发表文章进行比较。将不同模型的最终相对表达值以及中间输出结果与原始论文中的预期结果进行核对，发现其准确可靠。
pcr包为其主要功能提供了直观且统一的界面，使生物学家能够执行qPCR分析的所有必要步骤，并以统一的方式生成图表。
