Optimisation of sample preparation from primary mouse tissue to maintain RNA integrity for methods examining translational control

Abstract

AbstractProtein output of different mRNAs can vary by two orders of magnitude therefore it is critical to understand the processes that control gene expression that operate at the level of translation. Translatome-wide techniques such as polysome profiling and ribosome profiling are key methods for determining translations 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 needs to be avoided when requiring to maintain the interaction and position of the ribosome with the mRNA. 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.