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.
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.
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 heat2,3. 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 adipose tissue collection and handling are required4.
Among white adipose tissues, it is generally accepted that subcutaneous and visceral fat depots have different properties such as anatomical localization and function2,5. Consequently, to avoid conflicting results or large variability, attention needs to be taken to avoid cross-contamination between these different fat depots when collecting fat pads.
Moreover, there are three major challenges when isolating RNA or protein from mice white adipose tissue. First, collecting fat pads in obese mice is not an easy task as the border which separates different white fat depots is not always clear, in contrast to other organs such as the kidneys and heart6. Second, because of the high lipid content of adipose tissue, during RNA or protein isolation, a layer of lipids floats on top and prevents direct access to the sample. Third, as opposed to brown adipose tissue or other tissues, white adipose tissue has considerably lower RNA and protein content and this is of major concern when using young mice, mice fed a normal (N) diet and mice which are expected to have low fat masses (i.e. KO models, treatment with drugs, exercise training, etc.)7,8.
Therefore, choosing the appropriate method to isolate the RNA from adipose tissue is critical. Alternative methods to phenol/chloroform extraction are commercial kits. They are typically based on an initial phenol extraction step, followed by RNA purification on a column9. However, those kits are typically more expensive and give samples of lower yield, while the RNA quality might be variable but are less time consuming. However, one of the biggest advantages to phenol solution/chloroform extraction described here is the possibility of isolating RNA, DNA, and protein from a single sample10. Since mice fat pads are usually small and give small quantity of RNA and proteins (especially in lean mouse models), these protocols maximize the data one can get out of a small sample.
The objective of this paper is to describe in detail a method to ensure adequate sample collection from three mice white adipose tissue depots as well as quantity and quality of RNA isolation. RNA obtained by following this protocol can be used to perform real-time PCR assays. This protocol is not intended for RNA isolation from cultured primary adipocytes.
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
2. Preparation of Ground White Adipose Tissue for RNA Isolation
Note: Wear gloves for each step as skin contains RNases which can promote RNA degradation and as such, alters sample quality and RNA yield after isolation.
3. RNA Isolation from Ground Adipose Tissue
Caution: Phenol solution is harmful to the skin. Wear a lab coat, gloves, safety glasses and execute the procedure under a chemical hood. A step-by-step flowchart is shown in Figure 2.
4. Determination of Adipose Tissue RNA Concentration and Purity
5. Reverse-transcription (RT)
6. Real-time PCR for Gene Expression in Adipose Tissue
Note: Avoid exposing the fluorescent dye to light. The protocol below describes the amplification of the reference gene s1614. The primers and the PCR conditions should be modified according to the gene of interest.
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.
Before performing any experiments with the isolated RNA, its purity was evaluated as described in step 4.9. For each white adipose tissue, RNA isolation by phenol solution produced samples with adequate quality as the ratio OD260/OD280 was around 2.0 which is considered as pure for RNA (Table 2). Real-time PCR data showed that leptin mRNA expression was significantly increased in PGF, PRF, and SCF of obese mice compared to those on N diet (Figure 4A). Indeed, the differences observed in leptin mRNA expression were not due to a variation of s16, the reference gene used to normalize the results, between the two groups of mice as Ct values were not altered (Figure 4B). Thus, s16 can be reliably used as a reference gene for mRNA expression in PGF, PRF, and SCF when HF diet is a parameter in a study protocol.
Figure 1. Anatomical localization of white adipose tissue in mice. A male mouse on N diet has been dissected to show the localization of each adipose tissue depot and adrenal glands. SCF (A), PGF (B), PRF (C) and adrenal gland (D). PGF, peri-gonadal fat; PRF, peri-renal fat; SCF, abdominal subcutaneous fat. Please click here to view a larger version of this figure.
Figure 2. RNA isolation protocol flow chart using phenol solution. Schematic representation of the steps required to isolate RNA from grounded white adipose tissue. Please click here to view a larger version of this figure.
Figure 3. RNA quality assessment on bleach gel. 1 ug of RNA isolated from subcutaneous fat (SCF) and peri-gonadal fat (PGF) is separated on a 1% agarose bleach gel by electrophoresis. 28s and 18s rRNA are visualized by UV-transillumination. On gel are RNA isolated from SCF and PGF of a mouse fed a normal diet. Please click here to view a larger version of this figure.
Figure 4. Effect of HF diet on leptin and s16 mRNA expression in white adipose tissue. Leptin (A) and s16 (B) mRNA expression. Data for leptin are normalized to s16 mRNA levels while data for s16 are shown as Ct value and both are presented as mean ± SE with n = 12-13 per group. * p< 0.05 compared to N diet. N, normal; HF, high-fat; PGF, peri-gonadal fat; PRF, peri-renal fat; SCF, abdominal subcutaneous fat. This figure has been modified from Tan, P. et al, Obesity (2014) 22, 2201-2209. Please click here to view a larger version of this figure.
N diet | HF diet | |
Final body weight (g) | 37.5 ± 1.3 | 46.7 ± 1.7* |
Weight gain (g) | 6.6 ± 0.7 | 16.7 ± 1.0* |
PGF (g) | 1.08 ± 0.17 | 2.19 ± 0.15* |
PRF (g) | 0.79 ± 0.15 | 1.71 ± 0.06* |
SCF (g) | 1.62 ± 0.35 | 3.55 ± 0.20* |
Table 1. Effect of HF diet on body and white adipose tissue weights. Values are expressed as means ± SE with n = 14-15 per group. * p< 0.05 compared to N diet. N, normal; HF, high-fat; PGF, peri-gonadal fat; PRF, peri-renal fat; SCF, abdominal subcutaneous fat. This figure has been modified from Tan, P. et al, Obesity (2014) 22, 2201-2209.
Samples | Ratio OD260/OD280 |
PGF 1 | 2.04 |
PGF 2 | 2.04 |
PGF 3 | 2.02 |
PRF 1 | 1.95 |
PRF 2 | 2.08 |
PRF 3 | 2.08 |
SCF 1 | 2.04 |
SCF 2 | 2.02 |
SCF 3 | 2.06 |
Table 2. RNA purity after isolation from white adipose tissues. n = 3 per adipose tissue. PGF, peri-gonadal fat; PRF, peri-renal fat; SCF, abdominal subcutaneous fat.
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 contamination of one white adipose tissue depot with another as it is known that different adipose tissue depots have different functions2,5. Contamination can bring misleading results in downstream applications. In addition, as fat tends to dry once in contact with air, collecting white adipose tissue at a regular and consistent pace in between mice is recommended if the weight needs to be taken. Otherwise, the total weight of a fat pad might be incorrect. In order to get RNA of good quality and yield, following the recently published MIQE guidelines is a definite asset17. In particular, making very fine sample powder during grinding can help maximize yield. This maximizes the contact between the tissue powder and phenol solution during RNA isolation. Last but not least is the isolation of the RNA-containing layer during the RNA isolation (step 3.12). As fat is less dense than water, it is positioned on top of the phase of interest. Minimizing carry-over of fat is necessary to reduce interference with downstream applications.
One limitation of this method is the skill required to perform several steps in the procedure as well as the need to work with harmful reagents during RNA isolation. Moreover, the whole process is labor intensive.
There are not many alternatives to the method of adipose tissue collection and sample processing before RNA isolation apart from little details which are usually adjusted by each user. In the case of RNA isolation, many options are available such as the phenol/chloroform extraction or RNA isolation kits. There are advantages and disadvantages to each option and it is up to the user to select the best method based on downstream applications. Phenol/chloroform extraction is less expensive but requires the use of harmful reagents and is laborious. RNA isolation kits are generally more expensive but the procedure is faster and typically yields samples with good quality and purity. It is important to consider the yield and the purity of RNA because the material is limited in white adipose tissue which is mainly composed of fat. The major benefit in using phenol solution for isolation (phenol/chloroform extraction) is the possibility of isolating RNA, DNA, and protein from a single sample18. This is cost- and time-effective as it reduces the number of mice required to obtain sufficient material. As mentioned previously, fat mass is limited in some mouse models. For these mice, splitting ground adipose tissue into three parts to separately obtain RNA, DNA and protein is not recommended as this might lead to insufficient material for downstream applications. To circumvent this issue, it is also possible to pool together similar adipose tissue depot from several mice based on user-defined criteria, which leads to an increased number of animals needed.
The authors have nothing to disclose.
This work was supported by Diabetes Canada.
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 |