Nous remercions Jianhua Ling, Raymond MacDonald, Galvin Swift, Michelle Griffin, Paul Grippo et Satyanarayana Rachagani pour leurs commentaires utiles, des suggestions et le partage de leurs protocoles. Ce travail a été soutenu par U01CA111294 de subvention et un prix Idée de développement du Programme de recherche intra-muros Ohio State University.

1. Préparation Les pratiques suivantes adhèrent aux politiques établies par soin et l&#39;utilisation des animaux Comité de l&#39;institution (IACUC). <ol><li> Il est recommandé d&#39;isoler pas plus de 6 pancréas de souris en un jour donné. Vous avez tous les instruments chirurgicaux propres et autoclave, un jeu par animal. </li><li> Étiqueter tous les microtubes. Quatre tubes de 2 ml par animal. </li><li> Placer 8 ml de réactif de lyse dans 50 ml tubes de centrifugation sur la glace. Remarque: Bien que le kit de purification est livré avec un réactif au phénol de thiocyanate de guanidine qui est approprié pour l&#39;isolement du pancréas, le réactif de lyse est employé en raison de l&#39;importance des volumes qui sont nécessaires pour l&#39;isolation. </li><li> Distribuer environ 10 ml des solutions suivantes dans 15 ml tubes de centrifugation pour nettoyer les pales de l&#39;homogénéisateur. L&#39;éthanol, 70% (2 tubes), de l&#39;eau de qualité HPLC (1 tube) et de l&#39;eau de qualité HPLC contenant environ 200 pi de RNase extérieur ou ribonucléase inactivator équivalent. </li></ol&gt; 2. Chirurgie, Pancréas Dissection et homogénéisation <ol><li> Placez 1-2 ml d&#39;isoflurane dans un bocal contenant gaze de coton ou du papier absorbant. Les souris sont placées sur une plate-forme qui les empêche d&#39;atteindre la source de l&#39;isoflurane. Ajouter isoflurane frais dans le pot avant d&#39;anesthésier chaque souris. </li><li> Placez délicatement la souris dans le bocal contenant la isoflurane. Boucher le pot et surveiller les effets de l&#39;anesthésie en tournant doucement le pot avant et en arrière. L&#39;animal est anesthésié une fois qu&#39;il est dans l&#39;incapacité de se tenir dans sa &#39;propre. </li><li> Pour confirmation de l&#39;anesthésie, enlever l&#39;animal de la jarre et appliquer un pincement de l&#39;orteil entreprise. Si la souris ne réagit pas au pincement de l&#39;orteil, passez à l&#39;étape 2.5. </li><li> Si la souris réagit au pincement de l&#39;orteil, puis répétez les étapes 2.2 à 2.3. </li><li> Placez l&#39;animal anesthésié sur la paillasse en position couchée. Disloquer le col de la souris en plaçant bien la main gauche sur le col de la souris. Noustion de la main droite saisir la queue de la souris et rapidement tirer vers le haut à un angle d&#39;environ 45 ° à disloquer le col de l&#39;utérus. </li><li> Fixer la carcasse à une surface (un morceau de mousse de polystyrène recouvert de niveau avec des serviettes en papier) en perçant chacun des quatre pattes de l&#39;animal dans la surface plane en utilisant jauge 25 aiguilles hypodermiques. </li><li> Utilisation d&#39;instruments chirurgicaux stériles, couper la partie centrale de la souris et retirez le pancréas de la souris rapidement. Utilisez la pince pour tenir le pancréas et le couper de la rate et l&#39;intestin grêle avec des ciseaux à ressort. Veillez à ne pas étirer le pancréas lors de l&#39;enlèvement de la souris. Perturbation des tissus pourrait libérer ribonucléase. Remarque: Le temps qu&#39;il faut pour éliminer le pancréas de la souris est indispensable. Un homme du métier devrait être capable de disséquer le pancréas en environ 1 min ou moins. </li><li> Placer l&#39;ensemble du pancréas dans un tube de 50 ml contenant 8 ml de glace froid réactif de lyse. Boucher le tube et agiter brièvement à plonger le tiuestion dans le réactif et passer à l&#39;étape suivante sans retard. Le rapport de réactif de lyse de tissu est critique. Voir la section des résultats d&#39;une comparaison de l&#39;intégrité de l&#39;ARN isolé en utilisant différents volumes de réactif de lyse. </li><li> Homogénéiser pendant 5 sec. Il est important de presser les lames d&#39;homogénéisation au fond du tube de centrifugation pour permettre au tissu d&#39;être tiré dans les pales d&#39;homogénéisation efficace. </li></ol> 3. L&#39;élimination des tissus non digérés Note: Après l&#39;homogénéisation, de petits morceaux de tissu restent dans le réactif de lyse. Il est plus important pour lyser rapidement le tissu en laissant un peu de tissu non digéré plutôt que sur l&#39;homogénéisation et la dégradation des risques de l&#39;ARN. <ol><li> Transférer 2 ml de réactif de lyse contenant du pancréas lysées à un tube de microcentrifugation de 2 ml. Recueillir deux microtubes d&#39;homogénat par l&#39;isolement. Jeter le broyat ou magasin restant à -80 ° C pour de futures études. </li><li> Centrifuge à 4 ° C, 12 000 g pendant 5 min pour sédimenter les tissus non digérés. Il s&#39;agit d&#39;une étape essentielle dans le report des tissus non digérés dans les solutions suivantes de l&#39;ARN sera probablement contaminer l&#39;échantillon avec la ribonucléase (étape 5.13). </li><li> Transfert 1 ml du surnageant dans un tube de 2 ml à centrifuger. Procéder à l&#39;isolement immédiatement et stocker le tube répliqué à -80 ° C pour une utilisation future. Placer tous les tubes contenant homogénat sur de la glace tout en procédant à l&#39;étape suivante. </li><li> Éliminer le réactif de lyse restant dans un réceptacle de déchets chimique approprié. </li></ol> 4. Nettoyage des lames Homogénéisateur <ol><li> Avant de passer à la prochaine isolement, nettoyer les pales du homogénéisation en les plaçant dans 10 ml d&#39;éthanol à 70%. Activez l&#39;homogénéisation pendant environ 20 secondes pour enlever les morceaux de tissu qui peut être pris dans les pales. </li><li> Utiliser un embout de pipette 200 ul à enlever tous les morceaux qui restent dans les lames de homogénéisation. </li&gt; <li> Répétez l&#39;étape 4.1 dans l&#39;eau, inactivator général RNase et 70% d&#39;éthanol. Sécher l&#39;arbre d&#39;homogénéisation avec un chiffon propre Kim. </li><li> Répétez l&#39;étape 2.1 pour l&#39;isolement du pancréas des animaux restants, ARN garder isolés les uns séparés des animaux plutôt que la mise en commun de l&#39;ARN. </li></ol> 5. RNA La section suivante est identique au protocole de la trousse de purification. L&#39;avantage du kit de purification, c&#39;est qu&#39;il va isoler l&#39;ARN total, y compris les petits ARN. <ol><li> Si vous utilisez une solution de broyât congelé, décongeler sur la glace humide et laisser reposer à température ambiante pendant 5 min. </li><li> Ajouter 200 ul de chloroforme, boucher le tube et agiter vigoureusement à la main pendant 15 secondes. Laissez le tube reposer à température ambiante pendant 2-3 min. </li><li> Centrifuger pendant 15 min à 12 000 xg, 4 ° C. </li><li> Transférer la couche aqueuse supérieure dans un tube à centrifuger. Ajouter 1,5 volume d&#39;isopropanol et mélanger par pipetage de haut en bas plusieurs fois. </li><li> Transférer jusqu&#39;à 700 ul de l&#39;échantillon dans une colonne de centrifugation qui est placé dans un tube de collecte de 2 ml. Fermez le couvercle et centrifuger à 8000 g pendant 15 sec à température ambiante. </li><li> Jeter l&#39;effluent à travers. </li><li> Répéter les étapes 5.5 à 5.6, en utilisant le reste de l&#39;échantillon. </li><li> Ajouter 700 ul de tampon RTT à la colonne. Centrifuger pendant 15 s à 8000 x g. Jeter l&#39;effluent à travers. </li><li> Déposer 500 pi de tampon RPE dans la colonne. Centrifuger pendant 15 secondes à 8000 x g pour laver la colonne. Jeter l&#39;effluent à travers. </li><li> Ajouter 500 ul de tampon RPE à la colonne. Centrifuger pendant 2 min à 8000 xg pour sécher la colonne. </li><li> Transférer la colonne de centrifugation dans un tube de centrifugation de 1,5 ml. Pipette 80 pi d&#39;eau libre ribonucléase directement sur la membrane de la colonne. Centrifuger pendant 1 min à 8000 x g pour éluer l&#39;ARN. </li><li> Ajouter un inhibiteur de la RNase 1 ul de la solution d&#39;ARN. Stocker à -80 ° C ou procéder à 5.13. </li><li> Pour valider l&#39;intégrité de l&#39;ARN, d&#39;abord calculer la conce ARNntration et 260/280 taux à l&#39;aide d&#39;un spectrophotomètre. </li><li> Diluer une partie de la solution d&#39;ARN à environ 500 ng / ul avec de l&#39;eau de qualité biologie moléculaire. </li><li> Déterminer l&#39;ARN intégrité (c&#39;est-à-RIN) en utilisant une analyse par électrophorèse capillaire. Environ 5 ul de solution d&#39;ARN est suffisante pour l&#39;analyse. </li><li> Aliquoter la solution d&#39;ARN (voir la figure 2) et conserver à -80 ° C. </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.

Isolement de l&#39;ARN à partir de tissus qui sont ribonucléase riche représente un grand défi pour les expériences de biologie moléculaire. Différents réactifs et des kits sont disponibles dans le commerce qui sont principalement basées sur la méthode phénol thiocyanate de guanidine de l&#39;extraction de l&#39;ARN. Le thiocyanate de guanidine dénature les protéines et réduit l&#39;activité de la ribonucléase ainsi. Toutefois, l&#39;ampleur de la ribonucléase dans le pancréas nécessite des modificati...

Isolement de l&#39;ARN avec une haute intégrité est nécessaire pour des expériences de biologie moléculaire de routine telles que le nord-blot 9, qRT-PCR 1 ou profils d&#39;expression génique 5. La plupart des méthodes contemporaines de l&#39;isolement de l&#39;ARN sont fondées sur des modifications des protocoles de thiocyanate de guanidine 2, 3. Guanidine thiocyanate est un dénaturant de protéines forte et efficace de perturber l&#39;activité de la ribonucléase dans la plupart des conditions. La méthode populaire de Chomczynski et Sacchi 3 phénol combiné à la solution de thiocyanate de guanidine de lyse, ce qui réduit le temps d&#39;isolement à environ 4 heures. Disponibles dans le commerce de nombreux réactifs d&#39;extraction d&#39;ARN sont basées sur la méthode de Chomczynski et Sacchi 3. Ribonucléase tissus riches comme humain ou de souris pancréas présente un défi supplémentaire pour isoler l&#39;ARN. Pancréas de souris peut contenir jusqu&#39;à 75 mg de ribonucléase 6 dont certains seront publiés During perturbation du tissu pancréatique résultant en l&#39;autolyse des tissus. Des modifications des protocoles guanidine thiocyanate ont été utilisés avec succès pour isoler l&#39;ARN à partir du pancréas avec une grande intégrité de 2, 4, 7, 10. Infusion d&#39;ARN réactif de stabilisation dans le pancréas de souris isolement facilité de l&#39;ARN avec une haute intégrité 4, 7, 10, cependant infusion de solutions dans le pancréas nécessite une grande habileté, peuvent nécessiter des instruments spécialisés, tels que un microscope à dissection et une rupture de l&#39;architecture des tissus au cours de la procédure peut se traduire par la lyse cellulaire. Tissu perfusion avec le réactif de stabilisation de l&#39;ARN pourrait interférer avec d&#39;autres applications, y compris l&#39;isolement des protéines et coloration histologique. De plus, cette technique n&#39;est pas adaptée pour isoler l&#39;ARN à partir de pancréas humain. D&#39;autres protocoles nécessitent la préparation de solutions spécifiques par l&#39;enquêteur 2. Nous rapportons un protocole pour isoler l&#39;ARN avec une haute intégrité du pancréas de souris. Thprotocole e utilise des éléments de méthodes déjà publiées et repose en grande partie sur les protocoles réactifs standards phénol / guanidine thiocyanate base-lyse. Il ne nécessite pas la préparation de solutions spécialisées et ne nécessite pas la perfusion de réactifs dans le pancréas. Les étapes critiques pour l&#39;isolement de l&#39;ARN avec succès comprennent un phénol / à base de guanidine réactif de lyse rapport élevé de tissu, des prélèvements de tissus non digérée avant la séparation de phase et l&#39;inclusion d&#39;un inhibiteur de ribonucléase à la solution d&#39;ARN résultant. Utilisation de ceux-ci et d&#39;autres modifications, on isole l&#39;ARN avec RIN typiquement supérieur à 7 à utiliser en routine analyse de l&#39;expression génique.

<table border="0" cellpadding="0" cellspacing="0" width="788">
	<tbody>
		<tr height="21">
			<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>
		</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;">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>
		</tr>
		<tr height="42">
			<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>
		</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;">Rnase out (40 U/&#181;l)</td>
			<td style="width:93px;">Invitrogen</td>
			<td style="width:119px;">10777-019</td>
			<td>Rnase inhbitor</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="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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</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;">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>
		</tr>
		<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.

Le rendement total de l&#39;ARN à partir de 1 ml d&#39;homogénat de lyse est de 20 à 40 ug. DO 260/280 ratios sont généralement autour de 2,0 et le RIN sont constamment supérieur à 7,0. Si le RIN est ≤ 6, l&#39;isolement devra être répétée. De temps en temps, un RIN qui est supérieure à 8,0 est atteint. Les deux méthodes les plus couramment utilisées pour euthanasier les souris avant le retrait du pancréas sont le CO 2 asphyxie ou par inhalation d&#39;isoflurane....

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.

Isolement de l&#39;ARN de souris Pancréas: un tissu de Ribonuclease riche

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

Vidéo: 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.

Isolement des noyaux neuronaux à partir de tissus post-mortem du cerveau de l&#39;homme

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

Recherche

Biologie

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.

Isolation des myofibroblastes primaires de souris et de tissus du côlon humain

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.

In Vitro pancréas organogenèse du dispersées progéniteurs embryonnaires de souris

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.

Protein Isolation du développement embryonnaire Souris Coeur Région Valve

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.

Isolement et analyse fonctionnelle des mitochondries de cellules cultivées et de tissus de souris

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.

Isolation et culture des kératinocytes de la souris primaire de la peau de souris néonatale et adulte

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.

Isolation des noyaux par tissu entier à l’aide d’une méthode sans détergent et sans enzymes

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

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

Prélèvement de tissus et extraction d’ARN de l’articulation ostéoarthritique humaine du genou

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...

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.

Isolement de noyaux à partir de reins de souris adultes pour le séquençage de l’ARN mononucléaire

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

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Isolement, caractérisation et application thérapeutique de vésicules extracellulaires à partir de cellules souches mésenchymateuses humaines en culture

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...