Name: mouse adipose tissue collection and processing for rna analysis
Uploaded: 2018-01-31T14:00:00
Description: Watch this Scientific Journal Video about Mouse Adipose Tissue Collection and Processing for RNA Analysis at JoVE.com

This work was supported by Diabetes Canada.

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
<ol>
	<li>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...

<ol>
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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 ...

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.

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			<td height="21" style="height:21px;width:457px;">1 mL seringes</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">1X TE solution (10 mM Tris-HCl and 1 mM EDTA&#8226;Na2. pH 8.0)</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:457px;">22 G needles</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:457px;">26 G needles</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:457px;">75% Ethanol</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Block heater (dry bath)</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Chloroform</td>
			<td>Sigma</td>
			<td>C2432-500mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">dATP&#160;&#160;&#160;</td>
			<td style="width:189px;">Thermo scientific</td>
			<td style="width:189px;">R0141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">dCTP&#160;&#160;</td>
			<td style="width:189px;">Thermo scientific</td>
			<td style="width:189px;">R0151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">dGTP&#160;&#160;</td>
			<td style="width:189px;">Thermo scientific</td>
			<td style="width:189px;">R0161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">DNase I (1 U/&#181;l)&#160;</td>
			<td style="width:189px;">Thermo scientific</td>
			<td style="width:189px;">EN0521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">dTTP&#160;</td>
			<td style="width:189px;">Thermo scientific</td>
			<td style="width:189px;">R0171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Faststart Universal SYBR green Master (Rox)</td>
			<td style="width:189px;">Roche</td>
			<td style="width:189px;">4913922001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Faststart universal SYBR green master (Rox) fluorescent dye&#160;</td>
			<td style="width:189px;">Roche</td>
			<td style="width:189px;">4913914001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Filtered tips</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Forceps&#160;</td>
			<td style="width:189px;">Instrumentarium</td>
			<td>HB275</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Gauze</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Hammer</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">High fat rodent diet</td>
			<td>Bio-Serv, Frenchtown, NJ</td>
			<td style="width:189px;">F3282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Isopropanol&#160;</td>
			<td>Laboratoire MAT</td>
			<td>IH-0101</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:457px;">Leptin forward PCR primer (5&#8217;-GGGCTTCACCCCATTCTGA-3&#8217;) 10 uM</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:457px;">Leptin reverse PCR primer (5&#8217;-GGCTATCTGCAGCACATTTTG-3&#8217;) 10 uM</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Liquid nitrogen</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Maxima Reverse Transcriptase (enzyme and 5x buffer)</td>
			<td style="width:189px;">Thermo scientific</td>
			<td style="width:189px;">EP0742</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Nanopure water (referred as ultrapure water)</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Nitrile examination gloves</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Nitrile gloves</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:457px;">Normal rodent diet</td>
			<td style="width:189px;">Harlan Laboratories, Madison, WI</td>
			<td style="width:189px;">Harlan 2018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">P1000 pipetman</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">P2 pipetman</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:457px;">P20 pipetman</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:457px;">P200 pipetman</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Phenol solution (TRIzol)&#160;</td>
			<td style="width:189px;">Ambion Life Technologies</td>
			<td style="width:189px;">15598018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Pre-identified aluminium foil</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quartz spectrophotometer cuvette</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rack for PCR tube strips</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Racks for RT-PCR tube strips</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Random hexamers&#160;</td>
			<td style="width:189px;">Invitrogen</td>
			<td style="width:189px;">58875</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:457px;">Real-time PCR Rotor Gene system&#160;</td>
			<td style="width:189px;">Corbett research</td>
			<td style="width:189px;">RG-3000 Rotor-Gene thermal cycler</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Refrigerated bench-top centrifuge</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">RiboLock RNase Inhibitor&#160;</td>
			<td style="width:189px;">Thermo scientific</td>
			<td style="width:189px;">EO0381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">RNase-free 1.5 mL eppendorf tubes</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">RNase-free 1.5 mL screw cap tubes</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase-free PCR tube strips (0.2 mL) and caps</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">RNase-free water&#160;</td>
			<td style="width:189px;">Hyclone</td>
			<td style="width:189px;">SH30538.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">RT-PCR machine</td>
			<td style="width:189px;">Qiagen</td>
			<td style="width:189px;">Rotor-Gene Corbett 3000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">RT-PCR tube strips (0.1 mL) and caps</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:457px;">S16 forward PCR primer (5&#8217;-ATCTCAAAGGCCCTGGTAGC-3&#8217;) 10 uM</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:457px;">S16 reverse PCR primer (5&#8217; ACAAAGGTAAACCCCGATCG-3&#8217;) 10 uM</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Spectrophotometer</td>
			<td style="width:189px;">Biochrom</td>
			<td style="width:189px;">Ultrospec 3100 pro</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Stainless steel mortar and pestle</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="77">
			<td height="77" style="height:77px;width:457px;">Surgical pads</td>
			<td style="width:189px;">Home made a foam board wrapped in a disposable absorbent underpad</td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Surgical scissors&#160;</td>
			<td style="width:189px;">Intrumentarium</td>
			<td style="width:189px;">130.450.11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Thermal cycler</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Thermal cycler&#160;</td>
			<td style="width:189px;">Biometra</td>
			<td style="width:189px;">Thermocycler</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Vortex mixer</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Weighing spatula</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

mouse adipose tissue collection and processing for rna analysis

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.

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.
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Watch this Scientific Journal Video about Mouse Adipose Tissue Collection and Processing for RNA Analysis at JoVE.com

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.

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and ...

The overall goal of this procedure is to ensure adequate sample collection of white adipose tissue depots from different mice, and to isolate a high amount of good-quality RNA from the tissue. This method can answer key questions in the fields of metabolism, biochemistry and molecular biology that, for example, pertain to gene expression in adipose tissue from treated or genetically-modified mice. The main advantage of this technique is that it allows for the isolation of high amounts of good-quality RNA from adipose tissue which typically has low RNA content.Demonstrating the procedure will be Emilie Pepin, a research assistant from my laboratory. After euthanizing a mouse, cut the skin over the linea alba according to the text protocol, then make two incisions of approximately two centimeters from the middle of the ribcage towards the right arm and close to the genital organs above the right hind limb. Stretch one corner of the opened skin and use a 22-gauge needle to pin it down on the pad.Then, pin down the other corner. Using surgical scissors positioned against the skin as flat as possible, carry out blunt dissection of the abdominal subcutaneous fat, or SCF, then transfer it to a precut piece of aluminum foil. Repeat the dissection for the left side of the animal.Then, pull both the left and right collected fat pads in the aluminum foil and use liquid nitrogen to freeze the sample. To get access to the visceral adipose tissue, cut the abdominal muscle along the linea alba. Then, make an incision in the abdominal muscles from the genitals towards the back of the mouse on both sides.The fat around the reproductive organs is the perigonadal fat, or PGF. Detach the left and right fat pads from the epididymis, the testes and the ductus deferens and transfer it to the pre-labeled aluminum foil. Then, freeze the sample in liquid nitrogen.Starting with the left-hand side, expose the kidneys by moving the gut away from the abdominal cavity to the right-hand side. The adipose tissue seen here surrounding the kidney and the adrenal glands is the perirenal fat, or PRF. Isolate and remove the adrenal glands before collecting the PRF.This can be tedious as they are just a bit pinker than the adipose tissue. Now, isolate the PRF from the back muscular wall and cut the ureter, the renal artery and vein which separates the PRF and kidney from the rest of the body. Then, isolate the PRF from the kidney by cutting and removing the renal capsule.After isolating the right PRF, transfer the sample to the aluminum foil with the tissue from the left side and use liquid nitrogen to freeze the samples. Chill 1.5-milliliter RNase-free conical tubes, a mortar and pestle and a spatula in liquid nitrogen according to the text protocol. Put the adipose tissue sample in the mortar then use a few layers of brown paper to lift the pestle from the liquid nitrogen and fit it into the mortar.While holding the pestle with the paper, use the hammer to hit the top of the pestle once or twice. Next, grind the sample using a rotating motion until a fine powder is obtained. Then, remove the pestle, retrieve the spatula from the liquid nitrogen and use it to transfer the sample into the corresponding pre-chilled 1.5-milliliter conical tube.Dip the spatula back into liquid nitrogen from time to time to prevent the sample from melting. Also, keep the conical tube on dry ice to prevent the sample from thawing. Fill the conical tube to approximately 2/3 capacity with ground sample for adequate RNA yield.Then, prepare the remaining samples according to the text protocol. Under a chemical hood and while wearing a lab coat, gloves and safety glasses, remove a ground sample from liquid nitrogen. Slowly open the tube, and add 500 microliters of phenol solution.Close the lid of the tube and vortex at maximum speed for about five seconds, then slowly and carefully open the tube to release the pressure from the nitrogen. The sound of air coming out of the tube will be heard. Pipette an additional 500 microliters of phenol solution into the tube.Then, vortex and slowly open it as before. Vortex the sample until it is completely dissolved. Then, open the tube to release pressure before processing additional samples.Once all samples have been processed, briefly vortex them at maximum speed and incubate them at room temperature for five minutes. Centrifuge the tubes at 12, 000 times G and four degrees Celsius for 10 minutes. Then, bring the tubes back to room temperature.To avoid contamination, for each sample, use a P1000 pipette with a filtered tip to isolate as much RNA as possible from the middle pink layer by piercing the lipid phase near the side of the tube. Dispense the RNA into the new conical tubes without touching the sides of the tubes to prevent transferring lipids present on the outside of the tip. Add 200 microliters of pure chloroform to each sample and mix the tubes by inversion for 15 seconds.Then, after incubating the samples at room temperature for 10 minutes, centrifuge the tubes at 12, 000 times G and four degrees Celsius for 15 minutes and bring the tubes back to room temperature. For each sample, use a P1000 pipette and filtered tips to transfer as much aqueous transparent RNA-containing top phase as possible to the second set of corresponding conical tubes. Next, add 500 microliters of 100%isopropanol to each sample and mix the tubes by inversion for 15 seconds.Then, incubate the samples at room temperature for 10 minutes. Centrifuge the tubes at 12, 000 times G and four degrees Celsius for 10 minutes before bringing the tubes back to room temperature. Discard the isopropanol by pouring it out of each tube.Now, add one milliliter of 75%ethanol to each sample and briefly vortex the tubes at maximum speed. Then, centrifuge the samples at 7, 500 times G and four degrees Celsius for five minutes. After spinning the samples and bringing them to room temperature, discard the 75%ethanol by inverting each tube and lighting tapping them against brown paper to remove any residual ethanol.Leave the lid open and air-dry the RNA pellet for about 15 to 20 minutes. After evaluating the size of the RNA pellets, add 10 to 25 microliters of RNase-free water to each sample. Incubate the samples in a heat block at 60 degrees Celsius for five minutes.Then, briefly vortex the tubes. Incubate the samples in the same heat block for an additional five minutes. Then, quickly spin all the samples and immediately place them on ice.Carry out RT-PCR for gene expression in adipose tissue according to the text protocol. As expected, mice on the high fat diet had increased body weight and weight gain compared to litter mates on the normal diet. These observations were accompanied by a more than two-fold increase in the weight of the PGF, PRF and SCF in obese mice compared to those on the normal diet.This table shows that for each white adipose tissue, RNA isolation by phenol solution produced samples with an OD260 over OD280 of around 2.0 which is considered pure for RNA. As seen in this bar graph, realtime PCR data showed that leptin mRNA expression was significantly higher in PGF, PRF and SCF of obese mice compared to those on a normal diet. 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.Once mastered, this technique can be done in four hours if it is performed properly. While attempting this procedure, it's important to remember to work in an RNase-free environment during the RNA extraction procedure. Following this procedure, other methods like realtime PCR amplification can be performed in order to answer additional questions like gene expression modifications.After its development, this technique paved the way for researchers in the field of metabolism to explore adipose tissue modulations related to obesity and diabetes in rodents. After watching this video, you should have a good understanding of how to isolate different fat pads and isolate RNA from them. Don't forget that working with the phenol solution can be extremely hazardous, and precautions such as wearing a lab coat and gloves should always be taken while performing this procedure.

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