A Simple Protocol for High Efficiency Protein Isolation After RNA Isolation from Mouse Thyroid and Other Very Small Tissue Samples.
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.
Service of Endocrinology, Diabetology, and Metabolism, Lausanne University Hospital, Lausanne, Switzerland.
Panos G
PG
Ziros
Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA.
Dionysios V
DV
Chartoumpekis
Service of Endocrinology, Diabetology, and Metabolism, Lausanne University Hospital, Lausanne, Switzerland. gerasimos.sykiotis@chuv.ch.|Faculty of Biology and Medicine, University of Lausanne, Bureau BU44/07/2312, Rue du Bugnon 44, 1011, Lausanne, Switzerland. gerasimos.sykiotis@chuv.ch.
Gerasimos P
GP
Sykiotis
D000818:Animals; D019791:Guanidine / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D000074702:Proteostasis / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D013961:Thyroid Gland / Q000378:metabolism*
D035683:MicroRNAs; D011506:Proteins; D012333:RNA, Messenger; D019791:Guanidine
Guanidinium; Isolation; Micro-RNA; Proteostasis; QIAzol; RNA and protein; Simultaneous; TRI reagent; TRIzol; Thyroid