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A Simple Protocol for High Efficiency Protein Isolation After RNA Isolation from Mouse Thyroid and Other Very Small
Panos G Ziros1, Dionysios V Chartoumpekis2, Gerasimos P Sykiotis3,4
1Service of Endocrinology, Diabetology, and Metabolism, Lausanne University Hospital, Lausanne, Switzerland.
Methods in Molecular Biology (Clifton, N.J.)
|September 11, 2016
Summary
Researchers developed a new protocol to efficiently isolate RNA and protein from mouse thyroid tissue. This method enables detailed proteostasis studies in mice, overcoming previous limitations with small tissue size.
Area of Science:
- Molecular Biology
- Physiology
- Biochemistry
Background:
- The thyroid gland is crucial for hormone secretion and maintains proteostasis through various cellular responses.
- Research on thyroid proteostasis has predominantly used rats, limiting the application of mouse genetic models due to the small thyroid gland size.
- The small size of the mouse thyroid (~2 mg total) presents challenges for molecular and protein analysis.
Purpose of the Study:
- To present a simple, efficient protocol for simultaneous isolation of mRNA, micro-RNA, and protein from mouse thyroid tissue.
- To enable proteomic and transcriptomic studies in mouse thyroid using limited tissue samples.
- To facilitate the use of transgenic and knockout mouse models for thyroid proteostasis research.
Main Methods:
- Simultaneous isolation of mRNA, micro-RNA, and protein from ~1 mg of mouse thyroid tissue.
- A key modification addresses the solubilization of the protein pellet post-RNA extraction and precipitation.
- The protocol preserves one thyroid lobe for subsequent analyses like immunohistochemistry.
Main Results:
- Successfully isolated mRNA, micro-RNA, and 150-200 μg of protein from a single mouse thyroid lobe.
- Demonstrated the protocol's feasibility for downstream applications, including Western blotting.
- Validated the protocol's utility for comparative studies of proteostatic pathways in mouse thyroid and other tissues/cell lines.
Conclusions:
- The developed protocol overcomes the limitations of small mouse thyroid size for molecular analysis.
- This method supports the use of mouse models for comprehensive thyroid proteostasis research.
- The protocol is adaptable for various mouse tissues and rodent cell lines, broadening its applicability.
