Name: Video: Total RNA Isolation from Supraclavicular Brown Adipose Tissue for Gene Expression Analysis
Uploaded: 2024-03-29T14:10:00
Description: 161 Views. Total RNA Isolation from Supraclavicular Brown Adipose Tissue for Gene Expression Analysis

total rna isolation from supraclavicular brown adipose tissue for gene expression analysis

Morphological and Molecular Characterization of the scBAT in Mice

The increasing prevalence of obesity in the U.S. and worldwide has ignited great interest in understanding its etiology and identifying potential treatments1,2. Adipose tissue plays a vital role in metabolism, and dysregulation of the adipose tissue can lead to the development of obesity. Generally, there are two types of adipose tissues, white and brown adipose tissue. While white adipose tissue (WAT) can store chemical energy and secrete endocrine factors, brown adipose tissue (BAT) can use chemical energy to generate heat and maintain body temperature in the cold3,4. Because of this unique ability, activation of BAT can also increase energy expenditure and improve insulin sensitivity5.
BAT exerts its function through non-shivering thermogenesis, a process mediated by uncoupling protein 1 (UCP1)6. Mammals, including mice and humans, possess varying amounts of BAT. The classical view of BAT is that these adipose tissues are more abundant in mice and infants than in adult humans. iBAT, located in the upper dorsal flank between the scapulae, is the most studied BAT depot in mice. By applying radioisotope imaging and biopsy tests, recent studies identified several BAT depots in adult humans. Some of them, including the depots found in the deep neck and supraclavicular region, had not previously been identified in mice or other model animals7,8,9,10,11. Among these BAT depots, the scBAT is the most frequently seen depot in adult humans. To better understand the origin and molecular contribution of these newly found BAT depots in humans, it is essential to identify equivalent depots in mice that allow genetic and molecular manipulations to trace and test the functional role of these depots. Thus, we and others identified a few previously unknown BAT depots in different anatomical locations in mice, including scBAT12,13, thoracic perivascular BAT14,15, perirenal BAT16, and periaortic BAT17. Mouse scBAT anatomically resembles human scBAT and morphologically resembles classical iBAT, expressing high levels of UCP112.
Unlike mouse iBAT, which can be readily dissected, scBAT is situated in the intermediate layer of the mouse neck, beneath the salivary glands and along the external jugular vein. Isolation of this depot for histological and molecular analyses can be challenging. Here, we describe in detail the procedure for dissecting scBAT from postnatal and adult mice and processing this depot for histology and gene expression analysis.

Author Spotlight: Optimizing Supraclavicular Brown Adipose Tissue Extraction for Genetic Analysis

Dissection, Histological Processing, and Gene Expression Analysis of Murine Supraclavicular Brown Adipose Tissue

Total RNA Isolation from Supraclavicular Brown Adipose Tissue for Gene Expression Analysis

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Research

JoVE Journal

Biology

161 Views. Total RNA Isolation from Supraclavicular Brown Adipose Tissue for Gene Expression Analysis

Video: Total RNA Isolation from Supraclavicular Brown Adipose Tissue for Gene Expression Analysis

Brown adipose tissue (BAT)-mediated thermogenesis plays an important role in the regulation of metabolism, and its morphology and function can be greatly impacted by environmental stimuli in mice and humans. Currently, murine interscapular BAT (iBAT), which is located between two scapulae in the upper dorsal flank of mice, is the main BAT depot used by research laboratories to study BAT function. Recently, a few previously unknown BAT depots were identified in mice, including one analogous to human supraclavicular brown adipose tissue. Unlike iBAT, murine supraclavicular brown adipose tissue (scBAT) is situated in the intermediate layer of the neck and thus cannot be accessed as readily.
To facilitate the study of newly identified mouse scBAT, presented herein is a protocol detailing the steps to dissect intact scBAT from postnatal and adult mice. Due to scBAT&#39;s small size relative to other adipose depots, procedures have been modified and optimized specifically for processing scBAT. Among these modifications is the use of a dissecting microscope during tissue collection to increase the precision and homogenization of frozen scBAT samples to raise the efficiency of subsequent qPCR analysis. With these optimizations, the identification of, morphological appearance of, and molecular characterization of the scBAT can be determined in mice.

Here, we provide a practical procedure for dissecting and performing histological and gene expression analyses of murine supraclavicular brown adipose tissue.

Brown adipose tissue (BAT)-mediated thermogenesis plays an important role in the regulation of metabolism, and its morphology and function can be greatly ...