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

SIMPLE SUMMARY: The rate by which the ribosome decodes an mRNA determines the final protein output of the mRNA. This can vary greatly between different mRNAs. Methods which allow accurate determination of translation rates of specific mRNAs require conditions that preserve native mRNA–ribosome interactions and thus cannot be denaturing-based. Complex tissues sometimes contain RNases that can breakdown the RNA. Here, we optimise methods in non-denaturing conditions enabling the application of methods that examine translational control to primary mouse tissue in an accurate and reproducible manner. ABSTRACT: 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.