Name: Mouse Adipose Tissue Collection and Processing for RNA Analysis (Video) | JoVE Journal
Uploaded: 2018-01-31T14:00:00.000+00:00
Duration: 11 min 13 s
Description: Learn to collect white adipose tissue from mice and extract high-quality RNA for gene expression studies. This method enables reliable analysis of metabolic processes in treated or genetically-modified animals.

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This work was supported by Diabetes Canada.

<ol><li>Guh, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+incidence+of+co-morbidities+related+to+obesity+and+overweight%3A+a+systematic+review+and+meta-analysis%5BTitle%5D)+AND+%22BMC.+Public+Health%22%5BJournal%5D)">The incidence of co-morbidities related to obesity and overweight: a systematic review and meta-analysis.</a> BMC. Public Health. 9, 88(2009).</li>
<li>Wronska, A., Kmiec, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+and+biochemical+characteristics+of+various+white+adipose+tissue+depots%5BTitle%5D)+AND+%22Acta+Physiol+%28Oxf%29%22%5BJournal%5D)">Structural and biochemical characteristics of various white adipose tissue depots.</a> Acta Physiol (Oxf). 205 (2), 194-208 (2012).</li>
<li>Cannon, B., Nedergaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brown+adipose+tissue%3A+function+and+physiological+significance%5BTitle%5D)+AND+%22Physiol+Rev%22%5BJournal%5D)">Brown adipose tissue: function and physiological significance.</a> Physiol Rev. 84 (1), 277-359 (2004).</li>
<li>Holland, N. T., Smith, M. T., Eskenazi, B., Bastaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biological+sample+collection+and+processing+for+molecular+epidemiological+studies%5BTitle%5D)+AND+%22Mutat+Res%22%5BJournal%5D)">Biological sample collection and processing for molecular epidemiological studies.</a> Mutat Res. 543 (3), 217-234 (2003).</li>
<li>Ibrahim, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subcutaneous+and+visceral+adipose+tissue%3A+structural+and+functional+differences%5BTitle%5D)+AND+%22Obes+Rev%22%5BJournal%5D)">Subcutaneous and visceral adipose tissue: structural and functional differences.</a> Obes Rev. 11 (1), 11-18 (2010).</li>
<li>Mann, A., Thompson, A., Robbins, N., Blomkalns, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Localization%2C+identification%2C+and+excision+of+murine+adipose+depots%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Localization, identification, and excision of murine adipose depots.</a> J Vis Exp. (94), (2014).</li>
<li>Hemmrich, K., Denecke, B., Paul, N. E., Hoffmeister, D., Pallua, N. <a target="_blank" href="https://www.researchgate.net/publication/246759856_RNA_Isolation_from_Adipose_Tissue_An_Optimized_Procedure_for_High_RNA_Yield_and_Integrity">RNA Isolation from Adipose Tissue: An Optimized Procedure for High RNA Yield and Integrity.</a> Laboratory Medicine. 41 (2), 104-106 (2010).</li>
<li>Cirera, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+efficient+method+for+isolation+of+total+RNA+from+adipose+tissue%5BTitle%5D)+AND+%22BMC+Res+Notes%22%5BJournal%5D)">Highly efficient method for isolation of total RNA from adipose tissue.</a> BMC Res Notes. 6, 472(2013).</li>
<li>Tan, S. C., Yiap, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA%2C+RNA%2C+and+protein+extraction%3A+the+past+and+the+present%5BTitle%5D)+AND+%22J+Biomed+Biotechnol%22%5BJournal%5D)">DNA, RNA, and protein extraction: the past and the present.</a> J Biomed Biotechnol. 2009, 574398(2009).</li>
<li>Sellin Jeffries, M. K., Kiss, A. J., Smith, A. W., Oris, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comparison+of+commercially-available+automated+and+manual+extraction+kits+for+the+isolation+of+total+RNA+from+small+tissue+samples%5BTitle%5D)+AND+%22BMC+Biotechnol%22%5BJournal%5D)">A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples.</a> BMC Biotechnol. 14, 94(2014).</li>
<li>Tan, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+the+prorenin%2Frenin+receptor+on+the+development+of+obesity+and+associated+cardiometabolic+risk+factors%5BTitle%5D)+AND+%22Obesity+%28Silver.+Spring%29%22%5BJournal%5D)">Impact of the prorenin/renin receptor on the development of obesity and associated cardiometabolic risk factors.</a> Obesity (Silver. Spring). 22 (10), 2201-2209 (2014).</li>
<li>Taylor, S., Wakem, M., Dijkman, G., Alsarraj, M., Nguyen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+practical+approach+to+RT-qPCR-Publishing+data+that+conform+to+the+MIQE+guidelines%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines.</a> Methods. 50 (4), 1-5 (2010).</li>
<li>Aranda, P. S., LaJoie, D. M., Jorcyk, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bleach+gel%3A+a+simple+agarose+gel+for+analyzing+RNA+quality%5BTitle%5D)+AND+%22Electrophoresis%22%5BJournal%5D)">Bleach gel: a simple agarose gel for analyzing RNA quality.</a> Electrophoresis. 33 (2), 366-369 (2012).</li>
<li>Mercure, C., Prescott, G., Lacombe, M. J., Silversides, D. W., Reudelhuber, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+increases+in+circulating+prorenin+are+not+associated+with+renal+or+cardiac+pathologies%5BTitle%5D)+AND+%22Hypertension%22%5BJournal%5D)">Chronic increases in circulating prorenin are not associated with renal or cardiac pathologies.</a> Hypertension. 53 (6), 1062-1069 (2009).</li>
<li>Dusaulcy, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adipose-specific+disruption+of+autotaxin+enhances+nutritional+fattening+and+reduces+plasma+lysophosphatidic+acid%5BTitle%5D)+AND+%22J.+Lipid+Res%22%5BJournal%5D)">Adipose-specific disruption of autotaxin enhances nutritional fattening and reduces plasma lysophosphatidic acid.</a> J. Lipid Res. 52 (6), 1247-1255 (2011).</li>
<li>Nakamura, K., Fuster, J. J., Walsh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adipokines%3A+a+link+between+obesity+and+cardiovascular+disease%5BTitle%5D)+AND+%22J+Cardiol%22%5BJournal%5D)">Adipokines: a link between obesity and cardiovascular disease.</a> J Cardiol. 63 (4), 250-259 (2014).</li>
<li>Taylor, S. C., Mrkusich, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+state+of+RT-quantitative+PCR%3A+firsthand+observations+of+implementation+of+minimum+information+for+the+publication+of+quantitative+real-time+PCR+experiments+%28MIQE%29%5BTitle%5D)+AND+%22J+Mol+Microbiol+Biotechnol%22%5BJournal%5D)">The state of RT-quantitative PCR: firsthand observations of implementation of minimum information for the publication of quantitative real-time PCR experiments (MIQE).</a> J Mol Microbiol Biotechnol. 24 (1), 46-52 (2014).</li>
<li>Hummon, A. B., Lim, S. R., Difilippantonio, M. J., Ried, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+solubilization+of+proteins+after+TRIzol+extraction+of+RNA+and+DNA+from+patient+material+following+prolonged+storage%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Isolation and solubilization of proteins after TRIzol extraction of RNA and DNA from patient material following prolonged storage.</a> Biotechniques. 42 (4), 467-470 (2007).</li>
</ol>

The authors have nothing to disclose.

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.

<table><tbody><tr><td>1 mL seringes</td><td></td><td></td><td></td></tr><tr><td>1X TE solution (10 mM Tris-HCl and 1 mM EDTA&amp;bull;Na&lt;sub&gt;2&lt;/sub&gt;. pH 8.0)</td><td></td><td></td><td></td></tr><tr><td>22 G needles</td><td></td><td></td><td></td></tr><tr><td>26 G needles</td><td></td><td></td><td></td></tr><tr><td>75% Ethanol</td><td></td><td></td><td></td></tr><tr><td>Block heater (dry bath)</td><td></td><td></td><td></td></tr><tr><td>Chloroform</td><td>Sigma</td><td>C2432-500mL</td><td></td></tr><tr><td>dATP&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Thermo scientific</td><td>R0141</td><td></td></tr><tr><td>dCTP&amp;nbsp;&amp;nbsp;</td><td>Thermo scientific</td><td>R0151</td><td></td></tr><tr><td>dGTP&amp;nbsp;&amp;nbsp;</td><td>Thermo scientific</td><td>R0161</td><td></td></tr><tr><td>DNase I (1 U/&amp;micro;l)&amp;nbsp;</td><td>Thermo scientific</td><td>EN0521</td><td></td></tr><tr><td>dTTP&amp;nbsp;</td><td>Thermo scientific</td><td>R0171</td><td></td></tr><tr><td>Faststart Universal SYBR green Master (Rox)</td><td>Roche</td><td>4913922001</td><td></td></tr><tr><td>Faststart universal SYBR green master (Rox) fluorescent dye&amp;nbsp;</td><td>Roche</td><td>4913914001</td><td></td></tr><tr><td>Filtered tips</td><td></td><td></td><td></td></tr><tr><td>Forceps&amp;nbsp;</td><td>Instrumentarium</td><td>HB275</td><td></td></tr><tr><td>Gauze</td><td></td><td></td><td></td></tr><tr><td>Hammer</td><td></td><td></td><td></td></tr><tr><td>High fat rodent diet</td><td>Bio-Serv, Frenchtown, NJ</td><td>F3282</td><td></td></tr><tr><td>Isopropanol&amp;nbsp;</td><td>Laboratoire MAT</td><td>IH-0101</td><td></td></tr><tr><td>Leptin forward PCR primer (5&amp;rsquo;-GGGCTTCACCCCATTCTGA-3&amp;rsquo;) 10 uM</td><td></td><td></td><td></td></tr><tr><td>Leptin reverse PCR primer (5&amp;rsquo;-GGCTATCTGCAGCACATTTTG-3&amp;rsquo;) 10 uM</td><td></td><td></td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>Maxima Reverse Transcriptase (enzyme and 5x buffer)</td><td>Thermo scientific</td><td>EP0742</td><td></td></tr><tr><td>Nanopure water (referred as ultrapure water)</td><td></td><td></td><td></td></tr><tr><td>Nitrile examination gloves</td><td></td><td></td><td></td></tr><tr><td>Nitrile gloves</td><td></td><td></td><td></td></tr><tr><td>Normal rodent diet</td><td>Harlan Laboratories, Madison, WI</td><td>Harlan 2018</td><td></td></tr><tr><td>P1000 pipetman</td><td></td><td></td><td></td></tr><tr><td>P2 pipetman</td><td></td><td></td><td></td></tr><tr><td>P20 pipetman</td><td></td><td></td><td></td></tr><tr><td>P200 pipetman</td><td></td><td></td><td></td></tr><tr><td>Phenol solution (TRIzol)&amp;nbsp;</td><td>Ambion Life Technologies</td><td>15598018</td><td></td></tr><tr><td>Pre-identified aluminium foil</td><td></td><td></td><td></td></tr><tr><td>Quartz spectrophotometer cuvette</td><td></td><td></td><td></td></tr><tr><td>Rack for PCR tube strips</td><td></td><td></td><td></td></tr><tr><td>Racks for RT-PCR tube strips</td><td></td><td></td><td></td></tr><tr><td>Random hexamers&amp;nbsp;</td><td>Invitrogen</td><td>58875</td><td></td></tr><tr><td>Real-time PCR Rotor Gene system&amp;nbsp;</td><td>Corbett research</td><td>RG-3000 Rotor-Gene thermal cycler</td><td></td></tr><tr><td>Refrigerated bench-top centrifuge</td><td></td><td></td><td></td></tr><tr><td>RiboLock RNase Inhibitor&amp;nbsp;</td><td>Thermo scientific</td><td>EO0381</td><td></td></tr><tr><td>RNase-free 1.5 mL eppendorf tubes</td><td></td><td></td><td></td></tr><tr><td>RNase-free 1.5 mL screw cap tubes</td><td></td><td></td><td></td></tr><tr><td>RNase-free PCR tube strips (0.2 mL) and caps</td><td></td><td></td><td></td></tr><tr><td>RNase-free water&amp;nbsp;</td><td>Hyclone</td><td>SH30538.02</td><td></td></tr><tr><td>RT-PCR machine</td><td>Qiagen</td><td>Rotor-Gene Corbett 3000</td><td></td></tr><tr><td>RT-PCR tube strips (0.1 mL) and caps</td><td></td><td></td><td></td></tr><tr><td>S16 forward PCR primer (5&amp;rsquo;-ATCTCAAAGGCCCTGGTAGC-3&amp;rsquo;) 10 uM</td><td></td><td></td><td></td></tr><tr><td>S16 reverse PCR primer (5&amp;rsquo; ACAAAGGTAAACCCCGATCG-3&amp;rsquo;) 10 uM</td><td></td><td></td><td></td></tr><tr><td>Spectrophotometer</td><td>Biochrom</td><td>Ultrospec 3100 pro</td><td></td></tr><tr><td>Stainless steel mortar and pestle</td><td></td><td></td><td></td></tr><tr><td>Surgical pads</td><td>Home made a foam board wrapped in a disposable absorbent underpad</td><td></td><td></td></tr><tr><td>Surgical scissors&amp;nbsp;</td><td>Intrumentarium</td><td>130.450.11</td><td></td></tr><tr><td>Thermal cycler</td><td></td><td></td><td></td></tr><tr><td>Thermal cycler&amp;nbsp;</td><td>Biometra</td><td>Thermocycler</td><td></td></tr><tr><td>Vortex mixer</td><td></td><td></td><td></td></tr><tr><td>Weighing spatula</td><td></td><td></td><td></td></tr></tbody></table>

mouse adipose tissue collection and processing for rna analysis

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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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 ...

mouse-adipose-tissue-collection-and-processing-for-rna-analysis

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Learn to collect white adipose tissue from mice and extract high-quality RNA for gene expression studies. This method enables reliable analysis of metabolic processes in treated or genetically-modified animals.

Mouse Adipose Tissue Collection and Processing for RNA Analysis

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