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

1 Our lab is focused on establishing<v>2 supraclavicular brown adipose tissue 3 or SCBAT as a valuable depot 4 for brown fat analysis due to its anatomical similarities 5 to human brown adipose tissue. 6 This protocol is aimed at increasing accessibility<v>7 of SCBAT research. 8 This depot may be intimidating due to its small size 9 and proximity to large veins in the neck, 10 so it is imperative to provide 11 a simple method of extracting and processing SCBAT.12 Our methodology stands out<v>13 firstly by the use of a dissecting microscope 14 when extracting SCBAT. 15 This increases the precision 16 and extracting efficiency, 17 which are both critical when working 18 with such a small tissue depot. 19 Secondly, we homogenized the frozen SCBAT samples 20 before doing gene expression analysis 21 to increase the RNA yield to compensate 22 for such a low quantity of available tissue.23 We are currently focus on defining<v>24 the genetic lining edge of SCBAT, 25 identifying the transfiguration networks 26 regulating its development, 27 and developing a cerebral model 28 for exploring its physiological function in humans.

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

1 After dissecting scBAT<v>2 from the euthanized mouse, 3 immediately put the tissue into a micro centrifuge tube. 4 Poke a hole in the top of the tube with a sterile needle 5 and snap freeze in liquid nitrogen. 6 Soak a pestle and thin spatula in liquid nitrogen.7 Pour the snap frozen tissue sample 8 from the micro centrifuge tube into the mortar. 9 Add a small amount of liquid nitrogen to the mortar 10 and thoroughly grind the tissue with the pestle 11 until completely pulverized. 12 Use the thin spatula to scrape and transfer 13 the pulverized tissue back into the micro centrifuge tube.14 Once all tissue is transferred, 15 add 500 microliters of RNA isolation solution to the tube 16 and sonicate for 30 seconds using a pellet pestle motor. 17 Next, use a syringe to pipette up and down 10 times 18 to further lice the tissue, 19 ensuring no solid particles are visible. 20 Add 250 microliters of chloroform and vortex occasionally 21 during a five minute, room temperature incubation.22 Centrifuge the samples at 21, 000 G for 20 minutes 23 at four degrees Celsius. 24 Transfer the top aqueous layer into a new tube 25 and add an equivalent amount of 70%ethanol. 26 Transfer 500 microliters of the lysate 27 into a binding column.28 Centrifuge at 18, 000 G for 60 seconds 29 and discard the filtrate. 30 For reverse transcription, add 0.5 to one microgram 31 of RNA diluted in DEPC treated water to a PCR tube strip. 32 Then dilute the CDNA to 250 nanograms per microliter 33 using DEPC treated water and set up the quantitative PCR.34 The expression levels of marker genes involved 35 in mediating scBAT function were relatively similar 36 between scBAT and interscapular bat.

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JoVE Journal

Biology

127 Views. 1 After dissecting scBAT2 from the euthanized mouse, 3 immediately put the tissue into a micro centrifuge tube. 4 Poke a hole in the top of the tube with a sterile needle 5 and snap freeze in liquid nitrogen. 6 Soak a pestle and thin spatula in liquid nitrogen.7 Pour the snap frozen tissue sample 8 from the micro centrifuge tube into the mortar. 9 Add a small amount of liquid nitrogen to the mortar 10 and thoroughly grind the tissue with the pestle 11 until completely pulverized. 12...

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

dissection, histological processing, and gene expression analysis of murine supraclavicular brown adipose tissue

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

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isolating brown adipocytes from murine interscapular brown adipose tissue for gene and protein expression analysis

Isolating Brown Adipocytes from Murine Interscapular Brown Adipose Tissue for Gene and Protein Expression Analysis

This study describes a new method of isolating murine brown adipocytes for gene and protein expression...

dissection of supraclavicular brown adipose tissue in mice

Dissection of Supraclavicular Brown Adipose Tissue in Mice

supraclavicular brown adipose tissue processing for hematoxylin and eosin staining

Supraclavicular Brown Adipose Tissue Processing for Hematoxylin and Eosin Staining

isolation of total rna from pseudomonas aeruginosa within biofilms for measuring gene expression

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

This protocol presents a method to isolate RNA from Pseudomonas aeruginosa biofilms grown in chamber slides for high throughput...

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

rna isolation from embryonic zebrafish and cdna synthesis for gene expression analysis

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

The isolation of high quality, intact RNA is an essential step in many laboratory protocols.  Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray...

mouse adipose tissue collection and processing for rna analysis

Mouse Adipose Tissue Collection and Processing for RNA Analysis

The purpose of this paper is to present a step-by-step procedure to collect different white adipose tissues from mice, process the fat samples and extract RNA.

white and brown adipose depot collection from mouse pup: a surgical procedure to harvest the white adipose tissue and brown adipose tissue from mouse pup

In this video, we demonstrate the surgical procedure to isolate the subcutaneous white adipose tissue and interscapular brown adipose tissue from a newborn...

White and Brown Adipose Depot Collection from Mouse Pup: A Surgical Procedure to Harvest the White Adipose Tissue and Brown Adipose Tissue from Mouse Pup

To collect interscapular brown adipose tissue, pull the skin from the shoulder blades over the head, and lift the deep red brown adipose tissue located between the shoulder blades. Carefully make incisions all around the adipose to detach it from the body, and check the tissue for consistency and color. After rinsing, place the tissue in a second 1.5 milliliter tube containing 250 microliters of PBS and 200 microliters of 2X isolation buffer on...

chromatin immunoprecipitation of murine brown adipose tissue

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in...

isolation of viable adipocytes and stromal vascular fraction from human visceral adipose tissue suitable for rna analysis and macrophage phenotyping

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow...

isolation of nuclei from human intermuscular adipose tissue and downstream single-nuclei rna sequencing

Author Spotlight: Deciphering the Cellular Mysteries of Intermuscular Adipose Tissue in Humans

The biology of intermuscular adipose tissue (IMAT) is largely unexplored due to the limited accessibility of human tissue. Here, we present a detailed protocol for nuclei isolation and library preparation of frozen human IMAT for single nuclei RNA sequencing to identify the cellular composition of this unique adipose...

Isolation of Nuclei from Human Intermuscular Adipose Tissue and Downstream Single-Nuclei RNA Sequencing

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Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of...

single-cell nuclei isolation from homogenized human intermuscular adipose tissue samples for rna sequencing

Single-Cell Nuclei Isolation from Homogenized Human Intermuscular Adipose Tissue Samples for RNA Sequencing

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Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

cerenkov luminescence imaging of interscapular brown adipose tissue

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed...

Single Cell Nuclei Isolation from IMAT for Studying Gene Expression Profiles 

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infrared thermography for the detection of changes in brown adipose tissue activity

Infrared Thermography for the Detection of Changes in Brown Adipose Tissue Activity

Here, we present a protocol for measuring brown adipose tissue activity after a meal in humans and laboratory...

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 ...

1 To begin, place the mouse carcass<v>2 on the work bench. 3 Spray the ventral neck of the mouse with 70%ethanol 4 to minimize fur contamination, 5 make an approximately 1.5 centimeter long bilateral incision 6 along the clavicles. 7 From the ends of bilateral incisions, 8 cut about one centimeter longitudinally towards the ears, 9 creating a squared U-shaped incision around the neck.10 Place the mouse under a dissecting microscope 11 and bring the exposed neck into the focus. 12 Using forceps, lift the incised skin sections 13 to expose the salivary glands. 14 Now using surgical scissors 15 and forceps, disconnect the tissue connecting 16 the salivary glands to the surrounding clavicle area 17 and each other.18 Lift the salivary glands to expose 19 the supraclavicular brown adipose tissue or SC bat depots. 20 Observe the bat along the external jugular vein, 21 which is possibly connected to the underside 22 of the salivary glands. 23 Hold the target adipose tissue with forceps 24 and gently peel it away from the salivary glands.25 After removing adipose tissue, 26 continue detaching the SC bat from the external jugular vein 27 and the neck musculature until the deposes on either side 28 of the neck are fully removed. 29 Inspect the dissected SC bat sample 30 and use forceps to remove any remaining connective tissue.

1 Begin by placing<v>2 the glass scintillation vial containing 3 the mouse's scBAT in PBS onto the workbench. 4 Replace the PBS with 10 milliliters of cold, 5 4%paraformaldehyde. 6 Place the vial on a nutator, 7 rocking at four degrees Celsius overnight 8 to fix the scBAT.9 The next day, replace the paraformaldehyde solution 10 with 10 to 15 milliliters of PBS. 11 Place the vial on a nutator 12 and rock for 30 minutes at room temperature. 13 Replace the PBS with 0.85%saline 14 and continue rocking for another 30 minutes.15 Then replace the saline with 10 milliliters 16 of 70%ethanol, 0.85%saline solution. 17 Store the vial in a four degree refrigerator overnight 18 or until ready for paraffin embedding. 19 The next day, remove the 70%ethanol saline mixture 20 from the vial and add 10 to 15 milliliters 21 of 95%dehydrant alcohol.22 Rock the vial on a nutator at room temperature 23 for one hour. 24 After the second wash, add 10 to 15 milliliters 25 of 100%dehydrant alcohol 26 and continue rocking on a nutator for one hour. 27 To clear scBAT for embedding, add 10 milliliters of toluene 28 to the vial and continue rocking on a nutator 29 until scBAT is transparent.30 Then embed the scBAT in paraffin wax. 31 Section the scBAT with a microtome 32 and proceed for hematoxylin and eosin staining. 33 Hematoxylin and eosin staining confirmed 34 that scBAT comprises tissue structures characteristic 35 of brown adipose tissue, 36 including numerous small multilocular adipocytes 37 in both postnatal and adult mice.