Munro, J., Gillen, S. L., Mitchell, L., Laing, S., Karim, S. A., Rink, C. J., Waldron, J. A. and Bushell, M. (2023) Optimisation of sample preparation from primary mouse tissue to maintain RNA integrity for methods examining translational control. Cancers, 15(15), 3985. (doi: 10.3390/cancers15153985) (PMID:37568801) (PMCID:PMC10417042)
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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.
| Item Type: | Articles |
|---|---|
| Additional Information: | J.M., S.L.G., J.A.W. and M.B. were supported by Cancer Research UK core funding to the Beatson Institute (A31287) and to the Bushell Lab (A29252). L.M. was supported by Cancer Research UK core funding to the Beatson Institute (A31287). S.L. was supported by a CRUK Early Detection Project Award (C48702/A27603). S.A.K. and C.J.R. were supported by Cancer Research UK core funding to the Beatson Institute (A31287) and to the Morton Lab (A29996). C.J.R. was also supported by Pancreas Cancer Research UK Precision Panc grant (A25233). |
| Status: | Published |
| Refereed: | Yes |
| Glasgow Author(s) Enlighten ID: | Munro, Dr June and Karim, Mrs Saadia and Gillen, Miss Sarah and Waldron, Dr Joseph and Rink, Curtis and Laing, Dr Sarah and Bushell, Professor Martin and Mitchell, Mrs Louise |
| Authors: | Munro, J., Gillen, S. L., Mitchell, L., Laing, S., Karim, S. A., Rink, C. J., Waldron, J. A., and Bushell, M. |
| College/School: | College of Medical Veterinary and Life Sciences College of Medical Veterinary and Life Sciences > School of Cancer Sciences |
| Journal Name: | Cancers |
| Publisher: | MDPI |
| ISSN: | 2072-6694 |
| ISSN (Online): | 2072-6694 |
| Copyright Holders: | Copyright © 2023 The Authors |
| First Published: | First published in Cancers 15(15):3985 |
| Publisher Policy: | Reproduced under a Creative Commons License |
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