Quantification of the relative abundance of genetic traits has broad applications for biomarker discovery, diagnostics, and assessing gene expression in biological research. Relative quantification of genes is traditionally done with the 2^−ΔΔCT method using quantitative real-time polymerase chain reaction (qPCR) data, which is often limited in resolution beyond orders of magnitude difference. The latest techniques for quantification of nucleic acids employ digital PCR or microarrays which involve lengthy sample preparation and complex instrumentation. In this work, we describe a quantitative ratiometric regression PCR (qRR-PCR) method for computing relative abundance of genetic traits in a sample with high resolution from a single duplexed real-time quantitative PCR assay. Instead of comparing the individual cycle threshold (Ct) values as is done for the 2^−ΔΔCT method, our qRR-PCR algorithm leverages the innate relationship of co-amplified PCR targets to measure their relative quantities using characteristic curves derived from the normalized ratios of qPCR fluorescence curves. We demonstrate the utility of this technique for discriminating the fractional abundance of mixed alleles with resolution below 5%.

中文翻译：

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使用定量比率回归 PCR (qRR-PCR) 高分辨率估计相对基因丰度

遗传性状相对丰度的量化在生物研究中的生物标志物发现、诊断和评估基因表达方面具有广泛的应用。传统上，基因的相对定量是通过使用定量实时聚合酶链式反应 (qPCR) 数据的2 ^{−ΔΔCT方法来完成的，该方法的分辨率通常受到超出数量级差异的限制。}最新的核酸定量技术采用数字 PCR 或微阵列，这涉及冗长的样品制备和复杂的仪器。在这项工作中，我们描述了一种定量比率回归 PCR (qRR-PCR) 方法，用于通过单个双工实时定量 PCR 测定以高分辨率计算样品中遗传性状的相对丰度。我们的 qRR-PCR 算法不是像 2 ^−ΔΔCT方法那样比较单个循环阈值 (Ct) 值，而是利用共扩增 PCR 目标的固有关系，使用从归一化比率导出的特征曲线来测量其相对数量。 qPCR 荧光曲线。我们展示了该技术在区分混合等位基因的丰度分数方面的实用性，分辨率低于 5%。