Department of Morphology, Faculty of Veterinary Medicine, Ghent University, B-9820, Merelbeke, Belgium.
Department of Morphology, Faculty of Veterinary Medicine, Ghent University, B-9820, Merelbeke, Belgium; Department of Data Analysis and Mathematical Modelling, Ghent University, B-9000, Ghent, Belgium; Ghent University Digital PCR Consortium, Ghent University, B-9000, Ghent, Belgium.
Anal Biochem. 2021 Aug 1;626:114217. doi: 10.1016/j.ab.2021.114217. Epub 2021 May 1.
Accurate tools to measure RNA integrity are essential to obtain reliable gene expression data. The reverse transcription quantitative PCR (RT-qPCR) based 3':5' assay permits a direct determination of messenger RNA (mRNA) integrity. However, the use of standard curves and the possible effect of PCR inhibitors make this method cumbersome and prone to variation, especially in small samples. Here we developed a triplex digital PCR (dPCR) 3':5' assay for assessing RNA integrity in equine samples as rapid and simple alternative to RT-qPCR. This dPCR assay not only provides a straight forward analysis of the mRNA integrity, but also of its quantity.
准确的 RNA 完整性测量工具对于获得可靠的基因表达数据至关重要。基于反转录定量 PCR（RT-qPCR）的 3':5' 测定法允许直接测定信使 RNA（mRNA）的完整性。然而，标准曲线的使用和 PCR 抑制剂的可能影响使得这种方法繁琐且容易发生变化，尤其是在小样本中。在这里，我们开发了一种三重数字 PCR（dPCR）3':5' 测定法，作为 RT-qPCR 的快速简单替代方法，用于评估马样本中的 RNA 完整性。这种 dPCR 测定法不仅提供了对 mRNA 完整性的直接分析，还提供了其数量的分析。
