Mouse Adipose Tissue Collection and Processing for RNA Analysis

Summary

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.

Abstract

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and Western Blot, since it mostly contains lipids. RNA isolation from adipose tissue samples is also challenging as extra steps are required to avoid these lipids. Here, we present a procedure to collect three anatomically different white adipose tissues from mice, to process these samples and perform RNA isolation. We further describe the synthesis of cDNA and gene expression experiments using real-time PCR. The hereby described protocol allows the reduction of contamination from the animal's hair and blood on fat pads as well as cross-contamination between different fat pads during tissue collection. It has also been optimized to ensure adequate quantity and quality of the RNA extracted. This protocol can be widely applied to any mouse model where adipose tissue samples are required for routine experiments such as real-time PCR but is not intended for isolation from primary adipocytes cell culture.

Introduction

Obesity is a worldwide epidemic which can lead to complications such as type 2 diabetes¹. Diet-induced obese and genetically modified animal models are frequently used for research in obesity and its associated complications. Traditionally, white adipose tissue is known as a storage compartment for excess energy and is mostly composed of lipids while brown adipose tissue converts energy into heat^2,3. Adipose tissue is dynamic and will expand and shrink depending on many factors such as food intake and physical activity. Hence, to determine contributing factors to these changes, adequate....

Protocol

Care of the mice used in the procedures complied with standards for the care and use of experimental animals set by the Canadian Council for the Protection of Animals. All procedures were approved by the University Animal Care and Use Committee at the CHUM Research Center.

1. Necropsy and Adipose Tissue Collection from Male Mice

1. Make experimental groups according to study objectives. In this study, samples were collected from two groups of male mice on a C57Bl/6 background (n=12-14/group). Ensure that mice used here are 12-15 weeks of age and are maintained on 12-h light/dark cycle with access to either normal (N) or high-fat (H....

Results

Following the necropsy procedure, three white adipose tissues were collected and weighted from the two groups of mice (N and HF diet-fed mice). As expected, mice on the HF diet had increased final body weight and weight gain compared to littermates on N diet (Table 1). These observations were accompanied by more than a 2-fold increase in the weight of the PGF, PRF, and SCF in obese mice compared to those on N diet.

Discussion

Following HF-diet feeding, obese mice were found to have increased body and white adipose tissue weights compared to mice fed a N diet. RNA extracted using phenol solution yielded samples with good purity. Leptin is an adipokine primarily produced by adipose tissue and is known to correlate positively with fat mass¹⁶. As expected, leptin mRNA expression increased in obese mice in concomitance with their fat mass.

This method has several critical steps. The major one is .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL seringes
1X TE solution (10 mM Tris-HCl and 1 mM EDTA•Na2. pH 8.0)
22 G needles
26 G needles
75% Ethanol
Block heater (dry bath)
Chloroform	Sigma	C2432-500mL
dATP	Thermo scientific	R0141
dCTP	Thermo scientific	R0151
dGTP	Thermo scientific	R0161
DNase I (1 U/µl)	Thermo scientific	EN0521
dTTP	Thermo scientific	R0171
Faststart Universal SYBR green Master (Rox)	Roche	4913922001
Faststart universal SYBR green master (Rox) fluorescent dye	Roche	4913914001
Filtered tips
Forceps	Instrumentarium	HB275
Gauze
Hammer
High fat rodent diet	Bio-Serv, Frenchtown, NJ	F3282
Isopropanol	Laboratoire MAT	IH-0101
Leptin forward PCR primer (5’-GGGCTTCACCCCATTCTGA-3’) 10 uM
Leptin reverse PCR primer (5’-GGCTATCTGCAGCACATTTTG-3’) 10 uM
Liquid nitrogen
Maxima Reverse Transcriptase (enzyme and 5x buffer)	Thermo scientific	EP0742
Nanopure water (referred as ultrapure water)
Nitrile examination gloves
Nitrile gloves
Normal rodent diet	Harlan Laboratories, Madison, WI	Harlan 2018
P1000 pipetman
P2 pipetman
P20 pipetman
P200 pipetman
Phenol solution (TRIzol)	Ambion Life Technologies	15598018
Pre-identified aluminium foil
Quartz spectrophotometer cuvette
Rack for PCR tube strips
Racks for RT-PCR tube strips
Random hexamers	Invitrogen	58875
Real-time PCR Rotor Gene system	Corbett research	RG-3000 Rotor-Gene thermal cycler
Refrigerated bench-top centrifuge
RiboLock RNase Inhibitor	Thermo scientific	EO0381
RNase-free 1.5 mL eppendorf tubes
RNase-free 1.5 mL screw cap tubes
RNase-free PCR tube strips (0.2 mL) and caps
RNase-free water	Hyclone	SH30538.02
RT-PCR machine	Qiagen	Rotor-Gene Corbett 3000
RT-PCR tube strips (0.1 mL) and caps
S16 forward PCR primer (5’-ATCTCAAAGGCCCTGGTAGC-3’) 10 uM
S16 reverse PCR primer (5’ ACAAAGGTAAACCCCGATCG-3’) 10 uM
Spectrophotometer	Biochrom	Ultrospec 3100 pro
Stainless steel mortar and pestle
Surgical pads	Home made a foam board wrapped in a disposable absorbent underpad
Surgical scissors	Intrumentarium	130.450.11
Thermal cycler
Thermal cycler	Biometra	Thermocycler
Vortex mixer
Weighing spatula