Biz onların yararlı yorum, öneri ve protokol paylaşımı için Jianhua Ling, Raymond MacDonald, Galvin Swift, Michelle Griffin, Paul GRIPPO ve Satyanarayana Rachagani teşekkür ederim. Bu çalışma hibe U01CA111294 ve Ohio State Üniversitesi İntramural Araştırma Programı&#39;ndan bir Fikir Geliştirme Ödülü ile desteklenmiştir.

1.. Hazırlık Aşağıdaki uygulamalar Kurum&#39;un Hayvan Bakımı ve Kullanımı Komitesi (IACUC) tarafından belirlenen politikalara bağlı. <ol><li> Bu, herhangi bir gün içinde en fazla 6 fare pankreasları izole edilmesi tavsiye edilir. Temiz ve otoklav tüm cerrahi aletler, hayvan başına bir set var. </li><li> Tüm Mikrosantrifüj tüpleri etiketleyin. Hayvan başına dört 2 ml tüpler. </li><li> Buz üzerinde bir 50 ml santrifüj tüplerine reaktifi ile 8 ml yerleştirin. Not: Saflaştırma kiti pankreas izolasyonu için uygun olan bir guanidin tiyosiyanat fenol maddesi ile birlikte olsa da, liziz reaktif nedeniyle izolasyon başına gerekli olan büyük hacimlerde kullanılır. </li><li> Homojenleştirici ait bıçaklar temizlenmesi için bir 15 ml santrifüj tüplerine aşağıdaki çözümlerden yaklaşık 10 ml dağıtın. Etanol,% 70 (2 tüpler), HPLC dereceli su (1 tüp) yaklaşık 200 RNaz&#39;ın ul Dışarıda veya eşdeğer ribonükleez inaktivatör içeren HPLC dereceli su. </li></ol&gt; 2.. Cerrahi, Pankreas Diseksiyon ve Homojenleşmesi <ol><li> Pamuklu gazlı bez veya kağıt havlu içeren bir kavanoza izofluran 1-2 ml yerleştirin. Fareler izofluran kaynağını ulaşmasını engelleyen bir platform üzerine yerleştirilir. Öncesinde her fare anestezi için kavanoza taze isofluranın ekle. </li><li> Yavaşça izofluran içeren kavanozun içine fare yerleştirin. Kavanoz kapağı ve yavaşça ileri ve geri kavanoz çevirerek anestezi etkilerini izlemek. Onun kendi durmak mümkün olduğunu bir kez hayvan anestezi. </li><li> Anestezi teyidi için, kavanozdan hayvan kaldırmak ve sağlam bir ayak tutam uygulayın. Fare ayak tutam tepki vermezse, 2.5 adıma geçin. </li><li> Fare ayak tutam tepki, o adımları 2,2-2,3 tekrarlayın. </li><li> Yüzüstü pozisyonda tezgah üzerine anestezi hayvan yerleştirin. Sıkıca fare serviks üzerinde sol el koyarak mouse serviksini yerinden. Bizesağ fare kuyruğu kavramak ve hızlı bir şekilde rahim dağıtacağına yaklaşık yukarı bir 45 ° açı çekin ing. </li><li> 25 gauge derialtı iğneler kullanarak düz bir yüzeyin içine hayvanın dört pençeleri her delici bir yüzeye (kağıt havlu ile kaplı strafor bir seviye parça) için karkas sabitleyin. </li><li> Açık fare midsection kesti ve hızlı fare pankreas kaldırmak, steril cerrahi aletler kullanarak. Pankreas tutun ve yaylı makas kullanılarak dalak ve ince bağırsaktan kesmek için forseps kullanabilir. Fare çıkarılması sırasında pankreas germek için dikkatli olun. Doku bozulması Ribonuclease serbest olabilir. Not: Bu fare pankreas kaldırmak için gereken zamanı önemlidir. Yetenekli bir kişi, yaklaşık 1 dakika veya daha kısa bir pankreas incelemek gerekir. </li><li> Soğuk lisiz tepkin maddesinin 8 ml içeren 50 ml&#39;lik bir tüp içine tüm pankreas yerleştirin. Tüp kap ve kısaca ti öğrenmek sallamakayıraç içine ssue ve gecikmeden bir sonraki adıma geçin. Dokuya reaktifi oranı önemlidir. Liziz reaktifi farklı hacimleri kullanılarak izole RNA bütünlüğü bir karşılaştırma için sonuçlar bölümüne bakın. </li><li> 5 sn için homojenize edilmiştir. Bu doku etkili homojenizasyon için bıçakların içine çekilmesine izin vermek için, santrifüj tüpünün dibine homojenleştirici bıçakları basın önemlidir. </li></ol> Sindirilmemiş Doku 3. Kaldırma Not: Homojenizasyondan sonra doku küçük bit reaktifi kalacaktır. Bu hızlı homojenleştirici ve RNA risk bozulması üzerine yerine sindirilmemiş bazı doku bırakarak dokuyu parçalamak için daha önemlidir. <ol><li> Transfer 2 ml mikrosantrifüj tüpüne lize pankreas ihtiva eden liziz reaktifi 2 ml. Izolasyon başına homojenat iki mikrosantrifüj tüpleri toplayın. Gelecekteki çalışmalar için -80 ° C&#39;de kalan homojenatın veya mağaza atın. </li><li> Sent4 ° C, sindirilmemiş doku pellet haline getirmek için 5 dakika için 12,000 x g&#39;da rifuge. Bu büyük olasılıkla ribonüleaz (adım 5.13) ile numuneyi kontamine RNA sonraki çözümleri içine sindirilmemiş doku taşınmadan kaynaklanan gibi kritik bir adımdır. </li><li> 2 ml&#39;lik bir mikrosantrifüj tüpü içine aktarın süpernatan 1 ml. Hemen izolasyonu ile devam edin ve gelecekte kullanılmak üzere -80 ° C&#39;de suret tüp saklayın. Bir sonraki aşamaya geçmeden ıslak buz üzerinde Homojenat içeren tüm tüpler yerleştirin. </li><li> Uygun bir kimyasal atık yuvasına kalan reaktifi atınız. </li></ol> Homojenizetör Blades 4. Temizleme <ol><li> Bir sonraki izolasyon geçmeden önce,% 70 etanol, 10 ml yerleştirerek homojenleştirici bıçakları temizleyin. Bıçakları yakalanmış olabilir dokusunun herhangi parçalarını kaldırmak için 20 saniye boyunca yaklaşık için homojenizer etkinleştirin. </li><li> Homogenizer bıçakları kalan herhangi parçaları kaldırmak için bir 200 ul pipet ucu kullanın. </li&gt; <li> Suyu, genel Rnase inaktivatör ve% 70 etanol adımı tekrarlayın 4.1. Temiz Kim ile homojenleştirici şaft silin kurulayın. </li><li> Kalan hayvanların pankreas izolasyonu için yineleyin 2.1, yerine RNA havuzu daha her hayvan ayrı izole tutmak RNA. </li></ol> 5. RNA İzolasyon Aşağıdaki bölüm, saflaştırma kiti protokol ile aynıdır. Saflaştırma kiti avantajı küçük RNA&#39;ların de dahil olmak üzere, toplam RNA izole olmasıdır. <ol><li> Dondurulmuş homojenat çözeltisi kullanılarak ise, 5 dakika boyunca oda sıcaklığında beklemeye ıslak buz üzerinde ve çözülme sağlar. </li><li> Kloroform 200 ul ekleyin, tüp kap ve 15 saniye boyunca elle kuvvetlice sallayın. Tüp, 2-3 dakika boyunca oda sıcaklığında bekletin. </li><li> 12,000 xg, 4 ° C&#39;de 15 dakika boyunca santrifüj </li><li> Bir mikro santrifüj tüpüne üst Sulu tabakayı aktarın. Izopropanolün 1.5 sayılarını ve yukarı ve aşağı pipetleme birkaç kez iyice karıştırın. </li><li> 70 kadar transfer2 ml&#39;lik bir toplama tüpü içine yerleştirilen bir sıkma kolona numunenin 0 ul. Oda sıcaklığında 15 saniye boyunca 8000 x g&#39;de santrifüj kapağı ve kapatın. </li><li> Flow-through atın. </li><li> Tekrar, numunenin geri kalanı ile, 5,5-5,6 adımları. </li><li> Döndürme kolonuna 700 ul Tampon RWT ekleyin. 8,000 x g de 15 saniye boyunca santrifüjleyin. Flow-through atın. </li><li> Spin kolon içine pipet 500 ul Tampon RPE. Sütunu yıkamak için 8.000 xg 15 saniye santrifüj. Flow-through atın. </li><li> Döndürme kolonuna 500 ul Tampon RPE ekleyin. Sütunu kuru 8000 x g&#39;de 2 dakika boyunca santrifüjleyin. </li><li> Bir 1.5 ml mikrosantrifüj tüpüne spin kolon aktarın. Doğrudan spin kolon zar üzerine Pipet 80 ul ribonükleaz içermeyen su. RNA elüt edilmesi için 8000 x g &#39;de 1 dakika boyunca santrifüjleyin. </li><li> RNA çözeltisine, 1 ul Rnaz inhibitörü ekleyin. -80 ° C&#39;de saklayın veya 5,13 geçin. </li><li> RNA bütünlüğünü doğrulamak için, ilk olarak RNA conce hesaplamakBir spektrofotometre kullanılarak ntration ve 260/280 oranı. </li><li> Moleküler biyoloji dereceli su ile yaklaşık olarak 500 ng / ul RNA çözeltinin bir bölümünün seyreltin. </li><li> Kapiller elektroforez analizi kullanılarak RNA Dürüstlük (yani, Rin) belirleyin. RNA çözelti yaklaşık 5 ul analiz için yeterlidir. </li><li> -80 ° C&#39;de RNA çözeltisi (bkz. Şekil 2) ve mağaza alikotu </li></ol>

<ol>
	<li>Chen, C., Ridzon, D. A., Broomer, A. J., Zhou, Z., Lee, D. H., Nguyen, J. T., Barbisin, M., Xu, N. L., Mahuvakar, V. R., Andersen, M. R., 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=Real-time+quantification+of+microRNAs+by+stem-loop+RT-PCR.">Real-time quantification of microRNAs by stem-loop RT-PCR.</a> Nucleic Acids Res. 33, e179 (2005).</li><li>Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., Rutter, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+biologically+active+ribonucleic+acid+from+sources+enriched+in+ribonuclease.">Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.</a> Biochemistry. 18, 5294-5299 (1979).</li><li>Chomczynski, P., Sacchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-step+method+of+RNA+isolation+by+acid+guanidinium+thiocyanate-phenol-chloroform+extraction.">Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.</a> Anal Biochem. 162, 156-159 (1987).</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=Simplified+and+versatile+method+for+isolation+of+high-quality+RNA+from+pancreas.">Simplified and versatile method for isolation of high-quality RNA from pancreas.</a> Biotechniques. 52, 332-334 (2012).</li><li>Iyer, V. R., Eisen, M. B., Ross, D. T., Schuler, G., Moore, T., Lee, J. C., Trent, J. M., Staudt, L. M., Hudson Jr, J., Boguski, M. S., 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=The+transcriptional+program+in+the+response+of+human+fibroblasts+to+serum.">The transcriptional program in the response of human fibroblasts to serum.</a> Science. 283, 83-87 (1999).</li><li>Lenstra, J. A., Beintema, 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=The+amino+acid+sequence+of+mouse+pancreatic+ribonuclease.+Extremely+rapid+evolutionary+rates+of+the+myomorph+rodent+ribonucleases.">The amino acid sequence of mouse pancreatic ribonuclease. Extremely rapid evolutionary rates of the myomorph rodent ribonucleases.</a> European journal of biochemistry FEBS. 98, 399-408 (1979).</li><li>Mullin, A. E., Soukatcheva, G., Verchere, C. B., Chantler, J. 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, 617-621 (2006).</li><li>Rio, D. C., Ares, M., Hannon, G. J., Nilsen, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> RNA A Laboratory Manual. , (2011).</li><li>Streit, S., Michalski, C. W., Erkan, M., Kleeff, J., Friess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Northern+blot+analysis+for+detection+and+quantification+of+RNA+in+pancreatic+cancer+cells+and+tissues.">Northern blot analysis for detection and quantification of RNA in pancreatic cancer cells and tissues.</a> Nat Protoc. 4, 37-43 (2009).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> Journal of Visualized Experiments JoVE. , (2011).</li></ol>

The authors have nothing to disclose.

Zengin ribonükleaza olan dokulardan RNA izole moleküler biyoloji deneyleri için büyük bir sorun teşkil etmektedir. Çeşitli belirteçler, ve kitler öncelikle RNA ekstraksiyon fenol guanidin tiyosiyanat yöntemine bağlı ticari olarak temin dayanır ki. Guanidin tiyosiyanat protein denatüre ve böylece ribonükleaz etkinliğini azaltır. Ancak, pankreas ribonükleaz çokluğu bir RNA izolasyonu standart protokollere ek değişiklikler gerektirir. Standart bir protokol guanidin tiyosiyanat yöntemine göre oldu?...

Yüksek bütünlük RNA izolasyonu örneğin kuzey blot 9, qRT-PCR 1 ve 5 gen ekspresyonu gibi rutin moleküler biyoloji deneyleri için gereklidir. RNA izolasyonu çoğu modern yöntemler, guanidin tiyosiyanat protokolleri 2, modifikasyonları dayanmaktadır 3. Guanidin tiyosiyanat güçlü protein denatüran ve etkili bir çoğu durumda ribonükleaz aktivitesini bozacak. Chomczynski ve Sacchi 3&#39;ün yaygın kullanılan yöntem, yaklaşık 4 saat için izolasyon süresini azaltmak, guanidin tiyosiyanat lizis çözeltisine fenol birleştirildi. Pek çok ticari olarak temin edilebilen RNA ekstraksiyon reaktifleri Chomczynski ve Sacchi yöntem 3 üzerine dayanır. Bu tür insan veya fare gibi pankreas Ribonuclease zengin dokuların RNA&#39;nın izole edilmesi için ek bir zorluktur. Fare pankreas bazıları Durin çıkacak ribonükleaz 6 kadar olan 75 mg içerebilirg doku otoliz sonuçlanan pankreas dokusunun bozulması. Guanidin tiyosiyanat protokollerin modifikasyonları başarılı bir şekilde yüksek bütünlüğü 2, 4, 7, 10, yüksek bütünlüğü 4, 7, 10, RNA fare pankreası kolaylaştırılmış izolasyon içine RNA stabilizasyonu reajanı. Infusion Ancak infüzyon of ile pankreasından RNA&#39;yı izole etmek için kullanılmıştır pankreas içine çözümler, büyük bir beceri gerektirir böyle bir mikroskop ve işlem sırasında doku mimarisini bozan hücre parçalama neden olabilir gibi özel araçlar gerekebilir. RNA stabilizasyon reaktif ile dokuyu perfüze protein izolasyonu ve histolojik boyama gibi diğer uygulamaları ile etkileyebilir. Ayrıca, bu teknik, insan pankreasından RNA&#39;nın izole edilmesi için uygun değildir. Diğer protokoller 2 araştırmacı tarafından özel çözümler hazırlanmasını gerektirir. Biz fare pankreas yüksek bütünlük RNA izole etmek için bir protokol rapor. The protokolü daha önce yayınlanmış yöntemlerden elemanları içeren ve büyük ölçüde standart fenol / guanidin tiyosiyanat tabanlı liziz reaktif protokoller üzerine dayanır. Bu, özel bir çözeltilerin hazırlanmasını gerekli değildir, ne de pankreas içine reaktiflerin infüzyon gerektirir. Başarılı bir RNA izolasyonu için kritik adım doku oranı, elde edilen RNA çözeltiye ayırma ve bir ribonükleaz inhibitörü dahil faz önce sindirilmemiş doku çıkarılması için yüksek bir fenol / guanidin bazlı liziz reaktif içerir. Bu ve diğer değişiklikler kullanarak, tipik olarak daha büyük 7 rutin gen ekspresyon analizi için kullanılacak RIN ile RNA izole eder.

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			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:93px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:360px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Power Gen 500&#160;</td>
			<td style="width:93px;">Fisher Scientific</td>
			<td style="width:119px;">14-261-03&#160;</td>
			<td>Homogenizer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">5424R</td>
			<td style="width:93px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td>Refrigerated microcentifuge</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trizol reagent</td>
			<td style="width:93px;">Invitrogen</td>
			<td style="width:119px;">15596018</td>
			<td>Lysis reagent</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Student Vannas spring scissors</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;">91500-09&#160;</td>
			<td>use to cut pancreas from attached tissues</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical scissors, 6 inch</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;">08-951-20</td>
			<td>use to cut scin of mouse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blunt end forceps</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;">1381239</td>
			<td>use to hold pancreas while dissecting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Isoflurane USP</td>
			<td style="width:93px;">Abbott Labs</td>
			<td style="width:119px;">4/8/5210</td>
			<td>Anesthetic</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rnase out (40 U/&#181;l)</td>
			<td style="width:93px;">Invitrogen</td>
			<td style="width:119px;">10777-019</td>
			<td>Rnase inhbitor</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Rnase Away</td>
			<td style="width:93px;">Ambion</td>
			<td style="width:119px;">10328-011</td>
			<td>General Rnase inactivator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC grade water</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;">W5-4</td>
			<td>For washing homogenizer blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:216px;">Molecular Biology grade water</td>
			<td style="width:93px;">Hyclone</td>
			<td style="width:119px;">SH30538.03</td>
			<td>Elution of RNA</td>
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			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:216px;">miRNeasy Mini kit</td>
			<td style="width:93px;">Qiagen</td>
			<td style="width:119px;">217004</td>
			<td>Purification Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2 ml microcentrifuge tubes, certified Rnase Dnase free</td>
			<td style="width:93px;">USA Scientific Plastics</td>
			<td style="width:119px;">1620-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml centrifuge tubes</td>
			<td style="width:93px;">Falcon</td>
			<td style="width:119px;">352099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70% ethanol</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100% ethanol</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">200 ul pipet tips</td>
			<td style="width:93px;">Rainin</td>
			<td style="width:119px;">GPL200F</td>
			<td>For cleaning homogenizer blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;">BP1145-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nanodrop</td>
			<td style="width:93px;">Thermo</td>
			<td style="width:119px;">ND-1000</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bioanalyzer</td>
			<td style="width:93px;">Agilent</td>
			<td style="width:119px;"></td>
			<td>Capillary electrophoresis analyzer to measue RNA integrity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNAlater</td>
			<td style="width:93px;">Ambion</td>
			<td style="width:119px;"></td>
			<td>RNA Stabilization Reagent&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNeasy&#160;spin columns&#160;</td>
			<td style="width:93px;">Qiagen</td>
			<td style="width:119px;"></td>
			<td>&#160;spin columns&#160;as a part of the miRNeasy Mini kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mice</td>
			<td style="width:93px;">Chales River</td>
			<td style="width:119px;">strain C57BL/6</td>
			<td style="width:360px;">Their age ranged from 4 to 12 months.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mice&#160;</td>
			<td style="width:93px;">Jackson Lab</td>
			<td style="width:119px;">strain 129</td>
			<td style="width:360px;">Their age ranged from 4 to 12 months.&#160;&#160;</td>
		</tr>
	</tbody>
</table>

rna isolation from mouse pancreas: a ribonuclease-rich tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

Liziz Homojenat 1 ml toplam RNA verimi 20-40 ug&#39;dır. OD 260/280 oranı tipik olarak yaklaşık 2.0 &#39;dir ve RIN, 7.0&#39; dan, daha geniş bir bulunmaktadır. RIN 6 ≤ ise, izolasyon tekrar edilmesi gerekecektir. Bazen, 8.0 &#39;dan daha yüksek olan bir RIN elde edilir. Önce pankreas kaldırılması için fareler ötenazi iki en yaygın olarak kullanılan yöntemler, CO 2 asfiksi ya da izofluran teneffüs bulunmaktadır. Her iki teknik, servikal dislokasyon ile takip ed...

Watch this Scientific Journal Video about RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue at JoVE.com

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Fare pankreasından alınan RNA İzolasyonu: A Ribonuclease zengin Doku

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

rna-isolation-from-mouse-pancreas-a-ribonuclease-rich-tissue

Research

JoVE Journal

Biology

27.4K Görüntüleme.  The Ohio State University. We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Video: RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

İnsan Postmortem Beyin Dokusu Nöronal Çekirdekler İzolasyon

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Biyoloji

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

Fare ve İnsan Kolon Doku Primer miyofibroblastlar İzolasyonu

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

Dağınık Fare Embriyonik progenitörlerin gelen Vitro Pankreas organogeneziste

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Gelişmekte Embriyonik Fare Kalp Vana Bölgesi&#39;nde Protein İzolasyonu

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

İzolasyon ve Kültür Hücrelerinin ve Fare Doku gelen Mitokondri Fonksiyonel Analizi

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Yenidoğan ve Erişkin Fare Ciltlerindeki Primer Fare Keratinositlerin İzolasyonu ve Kültürü

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

Deterjan ve Enzimsiz YöntemLe Tüm Dokudan Çekirdek İzolasyon

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

Tek Çekirdekli RNA Dizilimi için Yetişkin Fare Böbreğinden Çekirdek İzolasyonu

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Plastoglobule Lipid Droplet Isolation from Plant Leaf Tissue and Cyanobacteria

Plastoglobule lipid droplets are a dynamic sub-compartment of plant chloroplasts and cyanobacteria. Found ubiquitously among photosynthetic species, they are believed to serve a central role in the adaptation and remodeling of the thylakoid membrane under rapidly changing environmental conditions. The capacity to isolate plastoglobules of high purity has greatly facilitated their study through proteomic, lipidomic, and other methodologies. With plastoglobules of high purity and yield, it is possible to investigate their lipid and protein composition, enzymatic activity, and protein topology, among other possible molecular characteristics. This article presents a rapid and effective protocol for the isolation of plastoglobules from chloroplasts of plant leaf tissue and presents methodological variations for the isolation of plastoglobules and related lipid droplet structures from maize leaves, the desiccated leaf tissue of the resurrection plant, Eragrostis nindensis, and the cyanobacterium, Synechocystis sp. PCC 6803. Isolation relies on the low density of these lipid-rich particles, which facilitates their purification by sucrose density flotation. This methodology will prove valuable in the study of plastoglobules from diverse species.

A fast and efficient protocol is presented for the isolation of plastoglobule lipid droplets associated with various photosynthetic organisms. The successful preparation of isolated plastoglobules is a crucial first step that precedes detailed molecular investigations such as proteomic and lipidomic analyses.

Plastoglobül Lipid Damlacığı İzolasyonu Bitki Yaprağı Dokusu ve Siyanobakterilerden

Plastoglobule lipid droplets are a dynamic sub-compartment of plant chloroplasts and cyanobacteria. Found ubiquitously among photosynthetic species, they ...

Fare Epididiminden Elde Edilen Yağ Dokusu Kaynaklı Kök Hücrelerin Primer Kültürü

Isolation, In Vitro Expansion, and Characterization of Mesenchymal Stem Cells from Mouse Epididymal Adipose Tissue

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential to modulate the immune response. The MSCs are immune-privileged cells capable of surviving in immunologically incompatible allogeneic transplant recipients based on low expression of class I major histocompatibility complex (MHC) molecules and in the use of cell-based therapy for allogeneic transplant. These cells can be isolated from several tissues, the most commonly used being the bone marrow and adipose tissues. We provide an easy protocol to isolate, culture, and characterize MSCs from epididymal adipose tissue of mice. The epididymal adipose tissue is surgically excised, physically fragmented, and digested with 0.15% collagenase type II solution. Then, primary adipose tissue-derived stem (ADSCs) cells are cultured and expanded in vitro, and the phenotypic characterization is performed by flow cytometry. We also provide the steps to differentiate the ADSCs into osteogenic, adipogenic, and chondrogenic cells, followed by functional characterization of each cell lineage. The protocol provided here can be used for in vivo and ex vivo experiments, and as an alternative, the adipose-derived stem cells can be used to generate MSCs-like immortalized cells.

Yazar Gündemi: Yağ Kaynaklı Kök Hücrelerle Tedavi Yaklaşımlarının Geliştirilmesi

The adipose tissue is an excellent source of mesenchymal stem cells. Here, we bring the step-by-step extraction, cultivation, and characterization of adipose tissue-derived stem cells (ADSCs) from Swiss mice epididymal adipose tissue.

Fare Epididimal Yağ Dokusundan Mezenkimal Kök Hücrelerin İzolasyonu, İn Vitro Genişletme ve Karakterizasyonu

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential ...