In the present study, two distinct PCR methods were used for the quantification of genetic material and their results were compared: real-time-PCR (qPCR; relative quantification) and droplet digital PCR (ddPCR; absolute quantification). The comparison of the qPCR and the ddPCR was based on a stimulation approach of microvascular endothelial cells in which the effect of a pro-inflammatory milieu on the expression of vasoactive receptors was investigated. There was consistency in directions of effects for the majority of genes tested. With regard to the indicated dimension of the effects, the overall picture was more differentiated. It was striking that deviations were more pronounced if the measured values were on the extreme edges of the dynamic range of the test procedures. To obtain valid and reliable results, dilution series are recommended, which should be carried out initially. In case of ddPCR the number of copies per µl should be adjusted to the low three-digit range. With regard to qPCR it is essential that the stability and reliability of the reference genes used is guaranteed. Here, ddPCR offers the advantage that housekeeping genes are not required. Furthermore, an absolute quantification of the sample can be easily performed by means of ddPCR. Before using ddPCR, however, care should be taken to optimize the experimental conditions. Strict indications for this methodology should also be made with regard to economic and timing factors.

中文翻译：

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相对和绝对RNA定量：基于血管活性受体内皮表达实例的比较分析

在本研究中，两种不同的PCR方法用于定量遗传物质，并比较了它们的结果：实时PCR（qPCR；相对定量）和液滴数字PCR（ddPCR；绝对定量）。qPCR和ddPCR的比较基于微血管内皮细胞的刺激方法，其中研究了促炎环境对血管活性受体表达的影响。大多数测试基因的作用方向一致。关于效果的指示尺寸，整体情况有所不同。令人惊讶的是，如果测量值位于测试程序动态范围的最边缘，则偏差会更加明显。为了获得有效和可靠的结果，建议使用稀释系列，应该首先进行。如果是ddPCR，则每微升的拷贝数应调整到较低的三位数范围。关于qPCR，必须确保所用参考基因​​的稳定性和可靠性。此处，ddPCR的优点是不需要管家基因。此外，可以通过ddPCR轻松地对样品进行绝对定量。但是，在使用ddPCR之前，应注意优化实验条件。还应在经济和时间因素方面对此方法进行严格说明。此处，ddPCR的优点是不需要管家基因。此外，可以通过ddPCR轻松地对样品进行绝对定量。但是，在使用ddPCR之前，应注意优化实验条件。还应在经济和时间因素方面对此方法进行严格说明。此处，ddPCR的优点是不需要管家基因。此外，可以通过ddPCR轻松地对样品进行绝对定量。但是，在使用ddPCR之前，应注意优化实验条件。还应在经济和时间因素方面对此方法进行严格说明。