小鼠和人类大脑样本及突触体制剂中信使核糖核酸的完整性检测
Integrity assay for messenger RNA in mouse and human brain samples and synaptosomal preparations.
作者信息
Bujanauskiene Daina, Merkevicius Kajus, Kuliesiute Ugne, Denkovskij Jaroslav, Kutanovas Simonas, Luksys Gediminas, Rocka Saulius, Bernotiene Eiva, Neniskyte Urte
机构信息
VU LSC-EMBL Partnership Institute for Genome Editing Technologies, Life Sciences Center, Vilnius University, Vilnius, Lithuania.
Institute of Biosciences, Life Sciences Center, Vilnius University, Vilnius, Lithuania.
出版信息
iScience. 2024 Jun 29;27(8):110419. doi: 10.1016/j.isci.2024.110419. eCollection 2024 Aug 16.
Traditionally, RNA integrity evaluation is based on ribosomal RNAs (rRNAs). Nevertheless, gene expression studies are usually focused on protein-coding messenger RNAs (mRNAs). Here, we present an RT-qPCR-based assay, which estimates mRNA integrity by comparing the abundance of 3' and 5' mRNA fragments. The assay was validated using plasmids with cloned 3'- and 5'-ends of the cDNA reflecting different ratios of 3' and 5' cDNA amplicons in partially degraded RNA samples. The accuracy of integrity value was ensured by including primer efficiency. We used 5':3' assay to quantify RNA degradation in heat- and enzyme-degraded mouse and human brain tissue RNA as well as in clinical human brain RNA samples. In addition, the 5':3' assay was suitable for assessing mRNA integrity in synaptosomal preparations that lack rRNAs. We concluded that the 5':3' assay can be used as a reliable method to evaluate mRNA integrity in tissue and subcellular preparations.
传统上，RNA完整性评估是基于核糖体RNA（rRNA）。然而，基因表达研究通常聚焦于蛋白质编码信使RNA（mRNA）。在此，我们提出一种基于逆转录定量聚合酶链反应（RT-qPCR）的检测方法，该方法通过比较3'和5'mRNA片段的丰度来估计mRNA完整性。使用含有克隆的cDNA 3'端和5'端的质粒对该检测方法进行了验证，这些质粒反映了部分降解的RNA样本中3'和5' cDNA扩增子的不同比例。通过纳入引物效率确保了完整性值的准确性。我们使用5':3'检测方法对热降解和酶降解的小鼠及人类脑组织RNA以及临床人类脑RNA样本中的RNA降解进行了定量。此外，5':3'检测方法适用于评估缺乏rRNA的突触体制剂中的mRNA完整性。我们得出结论，5':3'检测方法可作为评估组织和亚细胞制剂中mRNA完整性的可靠方法。
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