文章：

从原代小鼠组织中优化样本制备以维持RNA完整性用于研究翻译调控的方法

Optimisation of Sample Preparation from Primary Mouse Tissue to Maintain RNA Integrity for Methods Examining Translational Control

原文发布日期：5 August 2023

DOI: 10.3390/cancers15153985

类型: Article

开放获取: 是

英文摘要：

The protein output of different mRNAs can vary by two orders of magnitude; therefore, it is critical to understand the processes that control gene expression operating at the level of translation. Translatome-wide techniques, such as polysome profiling and ribosome profiling, are key methods for determining the translation rates occurring on specific mRNAs. These techniques are now widely used in cell lines; however, they are underutilised in tissues and cancer models. Ribonuclease (RNase) expression is often found to be higher in complex primary tissues in comparison to cell lines. Methods used to preserve RNA during lysis often use denaturing conditions, which need to be avoided when maintaining the interaction and position of the ribosome with the mRNA is required. Here, we detail the cell lysis conditions that produce high-quality RNA from several different tissues covering a range of endogenous RNase expression levels. We highlight the importance of RNA integrity for accurate determination of the global translation status of the cell as determined by polysome gradients and discuss key aspects to optimise for accurate assessment of the translatome from primary mouse tissue.

摘要翻译： 

不同mRNA的蛋白质产量可能相差两个数量级，因此，了解在翻译水平上控制基因表达的过程至关重要。全转录组范围的技术，如多聚体分析和核糖体分析，是确定特定mRNA上发生翻译速率的关键方法。这些技术目前已广泛应用于细胞系，但在组织和癌症模型中的应用相对不足。在复杂原代组织中，核糖核酸酶（RNase）的表达通常比细胞系更高。在裂解过程中用于保存RNA的方法通常采用变性条件，但在需要维持核糖体与mRNA的相互作用及其位置时，这些条件需要避免。在此，我们详细描述了从多种不同组织（涵盖一系列内源性RNase表达水平）中产生高质量RNA的细胞裂解条件。我们强调了RNA完整性对于通过多聚体梯度准确确定细胞整体翻译状态的重要性，并讨论了从原代小鼠组织中准确评估转录组的关键方面。

原文链接：

Optimisation of Sample Preparation from Primary Mouse Tissue to Maintain RNA Integrity for Methods Examining Translational Control