Name: rapid and cost-effective rna extraction of rat pancreatic tissue
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वर्तमान अध्ययन को शिराज आयुर्विज्ञान विश्वविद्यालय (अनुदान संख्या 93-01-01-7178\03-07-2014) द्वारा आर्थिक रूप से समर्थन दिया गया था । हम वीडियो के संपादन के लिए ई-लर्निंग इन मेडिकल साइंसेज, वर्चुअल स्कूल और सेंटर ऑफ एक्सीलेंस इन ई-लर्निंग, शिराज यूनिवर्सिटी ऑफ मेडिकल साइंसेज में श्री जोमोरोडियन और श्री रोस्टामी को धन्यवाद देते हैं ।

इस अध्ययन के लिए नैतिक अनुमोदन शिराज आयुर्विज्ञान विश्वविद्यालय (अनुमोदन संख्या: 93-01-01-7178\03-07-2014) से प्राप्त किया गया था ।
नोट: 250 ग्राम वजनी नर स्प्राग-डावले चूहों का उपयोग करें। -80 डिग्री से?...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+in+situ+ductal+perfusion+to+facilitate+isolation+of+high-quality+RNA+from+mouse+pancreas.">Application of in situ ductal perfusion to facilitate isolation of high-quality RNA from mouse pancreas.</a> Biotechniques. 40 (5), 617 (2006).</li><li>Li, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+method+using+TRIzol+reagent+and+liquid+nitrogen+produces+high-quality+RNA+from+rat+pancreas.">A modified method using TRIzol reagent and liquid nitrogen produces high-quality RNA from rat pancreas.</a> Applied Biochemistry and Biotechnology. 158 (2), 253-261 (2009).</li><li>Griffin, M., Abu-El-Haija, M., Abu-El-Haija, M., Rokhlina, T., Uc, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+and+versatile+method+for+obtaining+high+quality+rna+from+pancreas.">A simplified and versatile method for obtaining high quality rna from pancreas.</a> Biotechniques. 52 (5), 332 (2012).</li><li>Jun, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+optimization+for+extracting+high-quality+RNA+from+the+human+pancreas+tissue.">Method optimization for extracting high-quality RNA from the human pancreas tissue.</a> Translation Oncology. 11 (3), 800-807 (2018).</li><li>Green, M. R., Sambrook, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+win+the+battle+with+RNase.">How to win the battle with RNase.</a> Cold Spring Harbor Protocols. (2), 101857 (2019).</li><li>Li, D. -. S., Yuan, Y. -. H., Tu, H. -. J., Dai, L. -. j. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+islet+isolation+from+mouse+pancreas.">A protocol for islet isolation from mouse pancreas.</a> Nature Protocols. 4 (11), 1649 (2009).</li><li>Armstrong, J. A., Schulz, J. R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis.">Agarose gel electrophoresis.</a> Current Protocol: Essential Laboratory Techniques. (1), 1-20 (2008).</li><li>Aranda, P. S., LaJoie, D. M., Jorcyk, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleach+gel:+a+simple+agarose+gel+for+analyzing+RNA+quality.">Bleach gel: a simple agarose gel for analyzing RNA quality.</a> Electrophoresis. 33 (2), 366-369 (2012).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning:+a+laboratory+manual.">Molecular cloning: a laboratory manual.</a> Cold spring harbor laboratory press. , (1989).</li><li>Potenza, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybridase+activity+of+human+ribonuclease-1+revealed+by+a+real-time+fluorometric+assay.">Hybridase activity of human ribonuclease-1 revealed by a real-time fluorometric assay.</a> Nucleic Acids Research. 34 (10), 2906-2913 (2006).</li><li>Jackson, D., Lewis, F., Taylor, G., Boylston, A., Quirke, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+extraction+of+DNA+and+RNA+and+analysis+by+the+polymerase+chain+reaction.">Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.</a> Journal of Clinical Pathology. 43 (6), 499-504 (1990).</li><li>Quesada, I., Tudur&#237;, E., Ripoll, C., Nadal, &. #. 1. 9. 3. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+the+pancreatic+&#945;-cell+and+glucagon+secretion:+role+in+glucose+homeostasis+and+diabetes.">Physiology of the pancreatic &#945;-cell and glucagon secretion: role in glucose homeostasis and diabetes.</a> Journal of Endocrinology. 199 (1), 5-19 (2008).</li><li>Quertinmont, E., Nicaise, C., Gustot, T., Deviere, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Homogenization+with+the+MagNA+Lyser+Instrument+for+Total+RNA+Extraction+Using+the+TriPure+Reagent.">Tissue Homogenization with the MagNA Lyser Instrument for Total RNA Extraction Using the TriPure Reagent.</a> Liver (mg). 100 (100), 100 (2004).</li><li>Dastgheib, S., Irajie, C., Assaei, R., Koohpeima, F., Mokarram, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+RNA+extraction+from+rat+pancreatic+tissue.">Optimization of RNA extraction from rat pancreatic tissue.</a> Iranian Journal of Medical Sciences. 39 (3), 282 (2014).</li></ol>

आणविक जीव विज्ञान में उच्च गुणवत्ता वाले आरएनए प्राप्त करना महत्वपूर्ण है। कोशिकाओं और ऊतकों में राइबोन्यूक्लिज एंजाइमों की उपस्थिति जल्दी से आरएनए को नीचा दिखाती है और निष्कर्षण जटिल बनाती है। RNases ?...

संरचनात्मक जीन डेटा जीन अभिव्यक्ति के माध्यम से एक कार्यात्मक उत्पाद के लिए लिखित किया जा सकता है । आरएनए विश्लेषण का उपयोग विभिन्न परिस्थितियों में जीन अभिव्यक्ति में मतभेदों को खोजने के लिए किया जाता है। न्यूक्लियिक एसिड निकालने के लिए कई तरीके हैं: ग्वानिउद्दीनियम थिओसाइनेट, फिनोल-क्लोरोफॉर्म, सेल्यूलोज आधारित क्रोमेटोग्राफी के माध्यम से निष्कर्षण, सिलिका मैट्रिस द्वारा निष्कर्षण, और एनियन-एक्सचेंज1,,2।
जीन अभिव्यक्ति का उचित पता लगाना ऊतकों से अलग आरएनए की अखंडता से प्रभावित होता है; इसलिए, आगे के परीक्षणों से पहले ऊतकों से अलग आरएनए की अखंडता का मूल्यांकन करना महत्वपूर्ण है क्योंकि कम गुणवत्ता वाले आरएनए पर पूरक आणविक परीक्षण नैदानिक आवेदन परिणामों को ख़तरे में डालना हो सकता है। इस प्रकार, विभिन्न नैदानिक अनुप्रयोगों के साथ आणविक जैविक परीक्षणों के लिए उच्च अखंडता आरएनए की आवश्यकता होती है: मात्रात्मक आरटी-पीसीआर, माइक्रो-सरणी, राइबोन्यूलेज प्रोटेक्शन परख, उत्तरी दाग विश्लेषण, आरएनए मैपिंग, और सीडीएनए लाइब्रेरी निर्माण3,,4।
आरएनए एक लंबे समय के लिए रखा जा रहा है के बाद बल्कि अस्थिर हो जाता है । 10 केबी से अधिक लंबे एमआरएनए के टुकड़े विशेष रूप से5,6के क्षरण के लिए अतिसंवेदनशील होते हैं । इस प्रकार, शोधकर्ताओं को विभिन्न कारकों पर विचार करना चाहिए जो शुद्ध आरएनए की अखंडता को प्रभावित करते हैं। आरएनए की शुद्धता को आरएनएस, प्रोटीन, जीनोमिक डीएनए और एंजाइमेटिक अवरोधक संदूषण के खिलाफ संरक्षित किया जाना चाहिए। इसके अलावा, यूवी (260/280) के लिए आरएनए का सबसे अच्छा और स्वीकार्य अवशोषण अनुपात इलेक्ट्रोफोरेसिस पर न्यूनतम विखंडन के साथ 1.8-2.0 की सीमा के भीतर होना चाहिए । हाल ही में विकसित प्रयोगशाला तकनीकों ने वैज्ञानिकों को आणविक विश्लेषण नमूने की अखंडता का मूल्यांकन करने में सक्षम बनाया है , जो व्यावहारिक रूप से7,8
राइबोन्यूक्लिस (आरएनएस) की उच्च मात्रा के कारण अन्य प्रकार के ऊतकों की तुलना में अग्नाशय के ऊतकों से अक्षतिग्रस्त आरएनए निकालना बहुत अधिक कठिन है। हालांकि, मौजूदा निष्कर्षण विधियों, अर्थात् पेट की गुहा से अग्नाशय के ऊतकों का तेजी से इंजेक्शन और कम तापमान पर समरूपता आरनासेस को बाधित करने के लिए, अप्रभावी,,7,8,9,10, 11,,,,,12,1113, 14साबित हुए हैं।,14
वर्तमान तुलनात्मक प्रयोगात्मक अध्ययन का उद्देश्य सबसे कुशल तरीकों को निर्धारित करने के लिए मौजूदा तरीकों को संशोधित करना और तुलना करना है। इस उद्देश्य के लिए, आरएनए निष्कर्षण के विभिन्न प्रोटोकॉलों को संशोधित किया गया था और इसकी तुलना की गई थी। यह विशेष रूप से अग्नाशय के ऊतकों की एक ंयूनतम राशि की आवश्यकता होती है कम से कम महंगी विधि का निर्धारण करने के उद्देश्य से किया गया था।

<table>
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Merck</td>
			<td>116801</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Atoclave</td>
			<td>Teb Zaim</td>
			<td></td>
			<td>Iran</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sigma</td>
			<td></td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Merck</td>
			<td>107024</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Diethylpyrocarbonate (DEPC)-treated water</td>
			<td>Sigma</td>
			<td></td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>sigma</td>
			<td>60-00-4</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Electrophoresis tank</td>
			<td>Payapajoohesh</td>
			<td></td>
			<td>Iran</td>
		</tr>
		<tr>
			<td>Eppendorf microTube</td>
			<td>Extragene</td>
			<td></td>
			<td>Taiwan</td>
		</tr>
		<tr>
			<td>EtBr</td>
			<td>sigma</td>
			<td>E 8751</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>81870</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Falcon Tube</td>
			<td>Extragene</td>
			<td></td>
			<td>Taiwan</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Merck</td>
			<td>344198</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Merck</td>
			<td>344206</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Homogenizer-sunicator</td>
			<td>Microson XL 2000</td>
			<td></td>
			<td>USA</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>sigma</td>
			<td>19516</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td>sigma</td>
			<td>1867-66-9</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Laminar Flow Hood</td>
			<td>Jal Tajhiz</td>
			<td></td>
			<td>Iran</td>
		</tr>
		<tr>
			<td>Mgnetic stirrer</td>
			<td>Labrotechnik</td>
			<td></td>
			<td>USA</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Micropipette Tips</td>
			<td>Extragene</td>
			<td></td>
			<td>Taiwan</td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>sigma</td>
			<td>85022106</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Na AC</td>
			<td>Merck</td>
			<td>567422</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Merck</td>
			<td>109137</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Teb Zaim</td>
			<td></td>
			<td>Iran</td>
		</tr>
		<tr>
			<td>PH meter</td>
			<td>Knick</td>
			<td></td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>RNA Later/RNA stabilization reagent</td>
			<td>Qiagen</td>
			<td>76104</td>
			<td>USA</td>
		</tr>
		<tr>
			<td>Surgical instrument</td>
			<td>Agn Thos</td>
			<td></td>
			<td>German made</td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>AvaPezeshk</td>
			<td></td>
			<td>Iran</td>
		</tr>
		<tr>
			<td>TriPure reagent/RNA extraction reagent</td>
			<td>Roche</td>
			<td>11667157001</td>
			<td>USA</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Labinco</td>
			<td></td>
			<td>Netherland</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Memmert</td>
			<td></td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>zylazine</td>
			<td>sigma</td>
			<td>7361-61-7</td>
			<td>Germany</td>
		</tr>
	</tbody>
</table>

rapid and cost-effective rna extraction of rat pancreatic tissue

निष्कर्षण विधि के बावजूद, ऊतकों और सेल लाइनों का अनुकूलित आरएनए निष्कर्षण चार चरणों में किया जाता है: 1) समरूपता, 2) आरएनए से प्रोटीन का प्रभावी विकृति, 3) राइबोन्यूलाइज निष्क्रियता, और 4) डीएनए, प्रोटीन और कार्बोहाइड्रेट से संदूषण को हटाना। हालांकि, जब ऊतक में आरएनएएस के उच्च स्तर होते हैं तो आरएनए की अखंडता को बनाए रखना बहुत श्रमसाध्य होता है। सहज ऑटोलिसिस इसे नुकसान पहुंचाए बिना अग्नाशय के ऊतकों से आरएनए निकालने के लिए बहुत मुश्किल बना देता है। इस प्रकार, निष्कर्षण प्रक्रिया के दौरान अग्नाशय के ऊतकों की अखंडता को बनाए रखने के लिए एक व्यावहारिक आरएनए निष्कर्षण विधि की आवश्यकता होती है। मौजूदा प्रोटोकॉल का एक प्रयोगात्मक और तुलनात्मक अध्ययन 2 मिनट से भी कम समय में चूहा अग्नाशय के ऊतकों के 20-30 मिलीग्राम प्राप्त करने और आरएनए निकालने के द्वारा किया गया था । परिणामों का आकलन इलेक्ट्रोफोरेसिस द्वारा किया गया । परिणामों के सामान्यीकरण के लिए तीन बार प्रयोग किए गए । आरएनए स्थिरीकरण रिएजेंट में अग्नाशय के ऊतकों को विसर्जित करना -80 डिग्री सेल्सियस पर 24 एच के लिए उच्च अखंडता आरएनए, जब आरएनए निष्कर्षण अभिवाक्त्रक को रिएजेंट के रूप में इस्तेमाल किया गया था। प्राप्त परिणाम स्पिन कॉलम बाइंडिंग के साथ वाणिज्यिक किट से प्राप्त परिणामों के बराबर थे।

आरएनए स्टेबिलाइजेशन रिएजेंट के बिना एक नियमित और संशोधित सर्जिकल प्रोटोकॉल के अनुसार आरएनए निष्कर्षण रिएजेंट में आरएनए की अखंडता का मूल्यांकन एक नियमित शल्य चिकित्सा प्रोटोकॉल से आरएनए निष...

Watch this Scientific Journal Video about Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue at JoVE.com

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

अलग आरएनए की शुद्धता और अखंडता आरएनए निर्भर परख में एक महत्वपूर्ण कदम है । यहां, हम अक्षतिग्रस्त अग्नाशय के ऊतकों की एक छोटी मात्रा से आरएनए निकालने के लिए एक व्यावहारिक, तेजी से और सस्ती विधि पेश करते हैं।

चूहा अग्नाशय के ऊतकों की तेजी से और लागत प्रभावी आरएनए निष्कर्षण

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...

सभी को नमस्कार। हमने अग्नाशय के ऊतकों से कुशल आरएनए निष्कर्षण के लिए कक्षा वर्णनात्मक विधि में एक सीधी, तेजी से डिजाइन किया है। इस विधि का मुख्य लाभ ऑटोलिसिस के कारण ऊतक की न्यूनतम मात्रा से आरएनए निकालने के लिए एक अनुकूलित प्रक्रिया है। फिर से नमस्ते। प्रक्रिया का प्रदर्शन सनाज दस्ताग़ब द्वारा किया जाएगा, जो शिराज यूनिवर्सिटी ऑफ मेडिकल साइंसेज के बायोकेमिस्ट्री विभाग में हमारी प्रयोगशाला में मेरा पीएचडी छात्र है । पहले सर्जरी की जगह तैयार करें, सभी आवश्यकताओं को हुड के नीचे रखें । RNase को निष्क्रिय करने के लिए एक ओवन में सभी सर्जरी उपकरणों बंध्याकरण। केटामाइन-जाइलाज़ीन इंट्रापेरिटोनली 80/8 मिलीग्राम प्रति किलोग्राम इंजेक्ट करें। मौजूद ऊतकों में एंडोन्यूक्लियेज की सक्रियता में रहने के लिए उचित माइक्रोट्यूब में 1 cc.RNA स्थिरीकरण अभिवाक जोड़ें। तुरंत चूहे को सर्जिकल बोर्ड पर रखें। एक मिडलाइन चीरा के लिए सर्जन दूर सिर। फिर, चूहे के बालों को हटाने के लिए 70% शराब के साथ सभी पेट की सतह को स्टरलाइज करें। सर्जरी के लिए त्वचा को काटें। जघन क्षेत्र से सामने के पैरों तक पेट को खोलने के लिए चीरा को आकार दिया गया था। अग्न्याशय को बेनकाब करने के लिए पेट के अंगों को बाईं ओर फ्लिप करें। अग्न्याशय के ऊतकों को ध्यान से ढूंढें, जो पेट की गुहा में फैलता है। वसा ऊतक के साथ भ्रमित होने के बिना अग्न्याशय का पता लगाने के लिए प्रदर्शन के तहत क्षेत्र का पता लगाएं। दो मिनट से भी कम समय में पेट की गुहा से 20 से 30 मिलीग्राम अग्न्याशय तुरंत निकाल दें। हटाए गए ऊतकों को माइक्रो ट्यूब में बहुत जल्दी रखें और अलग ऊतकों के लिए आरएनए स्थिरीकरण अभिवाक में प्रवेश करने के लिए एस्ट्राइड कटर से काटें। माइक्रोट्यूब को तुरंत फ्रीज करने के लिए नाइट्रोजन टैंक में रखें। उनके सभी माइक्रोट्यूब 80 में रखे जाने चाहिए

rapid-and-cost-effective-rna-extraction-of-rat-pancreatic-tissue

Research

JoVE Journal

Biochemistry

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

मानव मैट्रल वाल्व से प्रोटीन के निष्कर्षण के लिए अनुकूलित प्रोटोकॉल

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

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.

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.

आरएनए विश्लेषण के लिए माउस वसा ऊतक संग्रह और प्रसंस्करण

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

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

स्तनधारी mRNAs पर अनुवाद दीक्षा परिसर गठन का Toeprinting विश्लेषण

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

HEK कोशिकाओं से Mitoribosome का तेजी से अलगाव

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

पर कब्जा और क्लिक करें रसायन-सहायता प्राप्त आरएनए-interactome कैप्चर (CARIC) रणनीति का उपयोग करके आरएनए बाइंडिंग प्रोटीन की पहचान

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Extraction and Analysis of Taiwanese Green Propolis

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and anticancer ones. The bioactive compounds of Taiwanese green propolis are propolins, namely C, D, F, and G. The concentration of propolins in Taiwanese green propolis varies depending on the season and geographic location. Thus, it is critical to establish a standard and repeatable procedure for determining the quality of Taiwanese green propolis. Here, we present a protocol that uses ethanol-based extraction, high-performance liquid chromatography, and an antibacterial activity analysis to characterize Taiwanese green propolis quality. This method indicates that 95% and 99.5% ethanol extractions achieve the maximum dry matter yields from Taiwanese green propolis, thereby yielding the highest concentrations of propolins that have antibacterial properties. According to these findings, the present protocol is deemed reliable and repeatable for determining the quality of Taiwanese green propolis.

We present a protocol for using ethanol as a solvent to extract and characterize Taiwanese green propolis that exhibits antibacterial activity.

निष्कर्षण और ताइवान ग्रीन एक प्रकार का पौधा का विश्लेषण

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

स्तनधारी कोशिका चक्र के दौरान सक्रिय प्रोटीन काइनेज आरएनए के आरएनए इंटरएक्टर्स का अध्ययन

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

4-Thiouracil (Ers4tU) के साथ Vivo में आरएनए की अत्यंत तेजी से और विशिष्ट मेटाबोलिक लेबलिंग

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

गैंग्लियोसाइड निष्कर्षण, शुद्धिकरण और प्रोफाइलिंग

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.