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

Author:

Munro June1,Gillen Sarah L.1,Mitchell Louise1,Laing Sarah2ORCID,Karim Saadia A.1,Rink Curtis J.12,Waldron Joseph A.1,Bushell Martin12

Affiliation:

1. Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, UK

2. School of Cancer Sciences, University of Glasgow, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1QH, UK

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.

Funder

Cancer Research UK

Publisher

MDPI AG

Subject

Cancer Research,Oncology

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