Relative versus absolute RNA quantification: a comparative analysis based on the example of endothelial expression of vasoactive receptors.
Relative versus absolute RNA quantification: a comparative analysis based on the example of endothelial expression of vasoactive receptors.
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DOI：
10.1186/s12575-021-00144-w
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发表时间：
2021-02-14
影响因子：
6.4
通讯作者：
Schumann J
中科院分区：
文献类型：
--
作者：
Kuhlmann K;Cieselski M;Schumann J
在本研究中，使用两种不同的 PCR 方法对遗传物质进行定量，并对它们的结果进行了比较：实时 PCR（qPCR；相对定量）和微滴数字 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是一致的。就所指出的效果维度而言，整体情况更加差异化。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，偏差会更加明显。为了获得有效和可靠的结果，建议首先进行系列稀释。如果是 ddPCR，每 µl 的拷贝数应调整至低三位数范围。对于 qPCR，保证所用参考基因的稳定性和可靠性至关重要。在这里，ddPCR 的优点是不需要管家基因。此外，通过 ddPCR 可以轻松地对样品进行绝对定量。然而，在使用 ddPCR 之前，应注意优化实验条件。该方法还应针对经济和时间因素做出严格说明。
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.
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影响因子：
3.1
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