TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span lang="EN-US" style="margin: 0px; line-height: 200%; font-family: 'Arial',sans-serif; font-size: 10pt;">ADAMTS5, the main aggrecanase</span><span lang="EN-US" style="margin: 0px; line-height: 200%; font-family: 'Arial',sans-serif; font-size: 10pt;">, is a protein that appears to play a functional role in development of white adipose tissue (WAT) and brown adipose tissue (BAT) and in browning of WAT. </span><span lang="EN-US" style="margin: 0px; line-height: 200%; font-family: 'Arial',sans-serif; font-size: 10pt;"> ADAMTS5 deficient or knockout (ADAMTS5<sup>-/-</sup> or KO) mice </span><span lang="EN-US" style="margin: 0px; line-height: 200%; font-family: 'Arial',sans-serif; font-size: 10pt;">indeed showed lower WAT mass, higher mass of interscapular BAT and enhanced browning of WAT, as indicated by elevated uncoupling-protein-1 (UCP-1) levels. </span><span lang="EN-US" style="margin: 0px; line-height: 200%; font-family: 'Arial',sans-serif; font-size: 10pt;">In addition, ADAMTS5<sup>-/-</sup> mice appeared to be protected from non-alcoholic steatohepatitis (NASH) when kept on a Western type diet</span><span lang="EN-US" style="margin: 0px; line-height: 200%; font-family: 'Arial',sans-serif; font-size: 10pt;">. These observations were made with the ADAMTS5-/--P mice originally generated by Pfizer, in collaboration with Lexicon Genetics. To provide confirmation of these observations, we have obtained a second strain of ADAMTS5 deficient mice, generated independently by Deltagen Inc.</span></p>

[BEFORE_START]
<p><em>All reagents (except DNAse) should be at RT; WORK QUICKLY@@</em></p>
<ul>
<li>Dry ice</li>
<li>2 ml collection tubes (1/sample)</li>
<li>1.5 ml Eppendorftubes (3/sample, 1 included in kit)</li>
<li>Pure ethanol</li>
<li>Ethanol (70% v/v) in RNAse free water (Molecular Biology Reagent W4502, Sigma-Aldrich, 35 ml ethanol + 15 ml water)</li>
<li>Qiagen Rneasy Mini kit (Catalog number 74106)</li>
<li>Qiagen RLT buffer: add 10 µl β-mercatoethanol (powder room, toxic: wear gloves and use hood) per 1 ml RLT buffer (stable for 1 month)</li>
<li>Qiagen RPE buffer (check if ethanol has been added)</li>
<li>RNAse-free DNAse I set (Qiagen: 79254): dissolve solid DNAse in 550 µl water provided in kit, <strong>DO NOT VORTEX, </strong>store at -20°C, thaw on ice. Make working solution: 10µl DNase stock solution (thaw on ice) + 70 µl RDD buffer (provided in kit) for each sample, mix by inverting (<strong>do not vortex</strong>). Perform on ice.</li>
<li>Clean bench first with dreft and then with ethanol</li>
<li>Clean eppendorf centrifuge: take out rotor, clean with dreft, rinse with hot water, rinse with ethanol and dry.</li>
<li>Write Lysing matrix D tubes (MP Biomedicals).</li>
</ul>

[GUIDELINES]
<p><span lang="EN-US" style="margin: 0px; line-height: 107%; font-family: 'Arial',sans-serif; font-size: 11pt;"><span lang="EN-US" style="margin: 0px; line-height: 107%; font-family: 'Arial',sans-serif; font-size: 12pt;">-RNA should be as pure as possible: OD<sub>260/280</sub> between 1.8 – 2.0 and OD<sub>260/230</sub> between 1.8 – 2.0. For sWAT and GON the OD<sub>260/230</sub> is sometimes lower than 1.8. Still these samples are used. <br /> -Volume of cDNA mix depends on the number genes of which the expression will be analyzed. E.g. 4 genes, 2µl/RT-PCR reaction, 2 technical replicates = 16µl; then 25µl is enough.</span></span></p>
<p><span lang="EN-US" style="margin: 0px; line-height: 107%; font-family: 'Arial',sans-serif; font-size: 11pt;">-Multiscribe enzyme: add enzyme last and keep it in a cold -20°C box.</span></p>

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