A Simple Protocol for High Efficiency Protein Isolation After RNA Isolation from Mouse Thyroid and Other Very Small Tissue Samples.

Details

Ressource 1Download: 27613051_AuthorManuscript.pdf (2226.48 [Ko])
State: Public
Version: Author's accepted manuscript
Serval ID
serval:BIB_F6CEDB51975B
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
A Simple Protocol for High Efficiency Protein Isolation After RNA Isolation from Mouse Thyroid and Other Very Small Tissue Samples.
Journal
Methods in molecular biology
Author(s)
Ziros P.G., Chartoumpekis D.V., Sykiotis G.P.
ISSN
1940-6029 (Electronic)
ISSN-L
1064-3745
Publication state
Published
Issued date
2016
Peer-reviewed
Oui
Volume
1449
Pages
383-393
Language
english
Notes
Publication types: Journal Article
Publication Status: ppublish
Abstract
As a dedicated hormone-secreting organ, the thyroid gland possesses a complement of proteostatic systems, including antioxidant, unfolded protein, and autophagic responses. The vast majority of animal investigations of thyroid physiology and, more recently, proteostasis, have utilized as model the rat, rather than the mouse. This is due to the very small size of the thyroid gland in the latter, with a total weight of ~2 mg (~1 mg per thyroid lobe). However, this strategy has limited the utilization of genetic approaches, such as taking advantage of the various transgenic and knockout mouse models. Here, we describe a simple and highly efficient protocol for the simultaneous isolation of mRNA, micro-RNA and 150-200 μg of protein from as little as 1 mg of mouse thyroid tissue, the average weight of one of the two thyroid lobes, thus preserving the other lobe for immunohistochemical or other analyses. While our workflow is similar to other protocols published in the literature and/or proposed by commercial reagent providers, we have introduced a key modification that addresses efficiently the most challenging step of the protein isolation process: the solubilization of the protein pellet after RNA extraction and protein precipitation. We demonstrate the feasibility of our approach and its utility for downstream analyses (including Western blotting) that facilitate the comparative study of proteostatic pathways in the mouse thyroid. We have also successfully applied this protocol on samples from mouse liver, brown and white adipose tissue, as well as from rodent cell lines.

Keywords
Animals, Guanidine/metabolism, Mice, MicroRNAs/metabolism, Proteins/chemistry, Proteins/isolation & purification, Proteostasis/physiology, RNA, Messenger/metabolism, Thyroid Gland/metabolism, Guanidinium, Isolation, Micro-RNA, Proteostasis, QIAzol, RNA and protein, Simultaneous, TRI reagent, TRIzol, Thyroid
Pubmed
Open Access
Yes
Create date
23/09/2016 18:43
Last modification date
20/08/2019 16:23
Usage data