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

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27.5K 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

Murine Pancreatic Islet Isolation

Hi, I am Greg Zott. I'm with the UCSF Diabetes Center in the Bluestone Laboratory, and today I'm gonna show you the process for isolating mouse eyelets. The procedure involves the injection of a collagenase solution into the pancreas of a C3 H donor mouse, where we will then remove that pancreas, digest it away, purify the eyelets on a fial gradient, and then handpick them for ultimate transplant into the kidney capsule of a diabetic NOD mouse, Which will be our recipient.Before we start this procedure, we diluted our ATE solution in a modified Hanks buffer and we have adjusted the concentration specific for the strain of mouse that we're using the C3 H.We found digests better at a 0.8 mgs per mil collagenase solution, and we will use that to inject into the common bile duct to distend the pancreas.Performing. The procedure today Will be Pavo codre of the diabetes center. He will inject, open up the mouse, expose the the intestines, and then inject into the common bile duct.We will spray down the mouse with ethanol to keep the hair down, and then we will also verify that the mouse has been sacrificed properly with a toe pinch. Pavo will now open up the mouse and exposing the intestine and the pancreas with a midline incision. The diaphragm will also be cut to verify that the mouse will be sacrificed.Before we get started, I'd like to point out some of the important structures. The stomach is lying right here with the pancreas attached underneath into the duodenum and above. This is the liver where we'll find the gallbladder and the common bile duct, which you can see right here.So what Pavo will do next is he will isolate the small intestine, find where the common bile duct enters the small intestine. Once the intestine is exposed, we clamp on either side of the common bile duct with mosquito clamps. We find that this allows more collagenates to enter the pancreas while other groups try to direct the clamp on top of the common bile duct.We find that this is a better technique in allowing enzyme to enter the pancreas. This is a gentle procedure. You don't want to tear the bowel.You wanna allow the clamps to lie gently on either side of the common bile duct. Now we will find a gallbladder, and once the gallbladder's exposed, we will use that as a guide to injecting the collagenase into the common bile duct. Powell's using a 30 gauge needle with a five cc syringe containing three mills of collagenase solution.As he's entering the common bile duct, he's distending that duct with collagenase solution and then he travels down the common bile duct till he reaches near the intestine. And as he's going down, the enzyme solutions being pumped into the injected into the pancreas. Now that the pancreas is inflated with collagenase solution, Powell's gonna remove the hemostats and then remove the pancreas from the the mouse carefully tearing it away from the intestine.He removes it from the stomach. Okay, and then using the spleen as a handle, he lifts up the pancreas and then slowly snips it away in the common bile duct, and that's it. Pancreas is laid out and then the spleen is removed, and there he's removing the meso enteric tissue, and then the pancreas is placed in a two mill collagenase solution and a sterilized glass vial and it's placed back on Ice.The isolated pancreas that we put into the glass vials will now be placed into a 37 degree water bath for approximately 17 minutes. We placed them into a 50 mil conical rack so that they don't float up. Once during the bath, the timer starts and the digestion begins.Once the digestion time is completed, vials are removed, and then they are shaken and disrupted gently. And once the tissue starts falling apart, the vial is then placed on ice. A sterile screen is placed over a 400 mil beaker that contains washing buffer.The pancreas digest is then carefully poured over the screen into the wash buffer. For demonstration purposes, the hood has been turned off and the glass of the hood has been raised so we could see what we're doing. Once the pancreas digest is poured into the beaker, the vials are rinsed with the same washing buffer and the wash buffer placed over the screen.We have a syringe filled with washing buffer. We will use that to forcefully wash the tissue through the screen. The idea is to release any loose eyelets that are unattached to the matrix, but leaving the membranes and the undigested tissue on the screen.And when you're finished washing a tissue, this is what you usually See at the end of the procedure. The Pancreas tissue that's in the bottom of the tube will now be transferred to two 50 mil conical, and the tissue will be washed, the tissue's mixed evenly, and then distributed evenly so that we have a equal pellet volume in each tube. The beaker is now rinsed with more washing buffer.The sides are rinse so that you get all tissue that is entered the beaker again. Then it's equally distributed between each two, both 50 mil. Conical then are topped off with wash buffer.The tissue is inverted several times and then placed into a centrifuge. This is a quick spin. The centrifuge is allowed to reach a thousand RPM and then is turned off.The tissue at this time is very S eyelets are very sensitive, and you don't want to over compact them. The tubes are removed and we will do this wash step three more times. This is the end of the third wash.The components of the 50 mil conical tube are transferred to the 15 conical tube and centrifuge for a thousand RP M and then stopped. The supernat will be removed and the first part of the FI Call will be started. We are at the step where we Have our pelleted di pancreas digest.We'll resuspend that tissue so it's nice and loose to the pellets. We'll add the first density gradient, which is 1 1 0 8. We'll add five mils to each tube.The pellet is then mixed thoroughly in each of the gradients. It's important that you get good tissue distribution in your heaviest gradient. Now we will layer two mils of 1 0 9 6 density.We will then on top of that, layer two mils of 1 0 6 9 density, and then on top of that 1 0 3 7 density. It's important while you're layering the densities that you don't mix. Then all the layers have been added to the tube.And you can see this is the top of 1 1 0 8, the top of 9 1 0 9 6, the top of 1 0 6 9 and the top of 1 0 3 7. And you could see the tissue is already starting to move to its proper density even without centri fusion. The fial gradients are brought up to speed at 1800 RPM for 15 minutes, and remember to keep the brake off after the tubes are removed from the centrifuge.You can see between the 0 0 9 6 and 0 0 7 layer, there's a nice band of eyelet tissue Pablo's. Now removing the 0 3 7 layer to remove any tissue debris and DNA that might have been released during the processing. Using a sterile plastic pasture pipette, he will now remove the islet layer and transfer them to a 50 mil conical containing 25 mils of wash buffer.You want do this step quickly because fol is toxic to eyelets. We actually removed the entire 0 6 9 and 0 9 6 layer. Pavo cuts the top of the plastic pasture pipette and rinses it with wash buffer.The eyelets are then centrifuge like we centrifuge previously. Centrifuge is brought up to a Thousand RPM and then we stop it and We aspirate off. The supernatant eyelets will be washed three times with wash media.So we just completed the eyelid Isolation process we took from the digest to the fol gradient to the purified eyelets after our third wash with wash buffer, we resuspended the eyelets in our RPMI media and we played it in a tissue culture plate. These eyelets are now ready for in vitro assays or in vivo preps like eyelet Transplant.

Isolement murin îlots pancréatiques

Recherche

Biologie

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

In Vitro Colony Assays pour Caractériser les cellules progénitrices Tri-puissants Isolée des adultes murins Pancréas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Biologie du développement

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

Isolation efficace des îlots fonctionnels de néonatales Pancréas souris

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

Séquençage et analyse de l'ARN unicellulaire des îlots pancréatiques humains

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Génétique

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective denaturation of proteins from RNA, 3) ribonuclease inactivation, and 4) removal of contamination from DNA, proteins, and carbohydrates. However, it is very laborious to maintain the integrity of RNA when there are high levels of RNase in the tissue. Spontaneous autolysis makes it very difficult to extract RNA from pancreatic tissue without damaging it. Thus, a practical RNA extraction method is needed to maintain the integrity of pancreatic tissues during the extraction process. An experimental and comparative study of existing protocols was carried out by obtaining 20-30 mg of rat pancreatic tissues in less than 2 minutes and extracting the RNA. The results were assessed by electrophoresis. The experiments were carried out three times for generalization of the results. Immersing pancreatic tissue in RNA stabilization reagent at -80 &#176;C for 24 h yielded high integrity RNA, when the RNA extraction reagent was used as the reagent. The results obtained were comparable to the results obtained from commercial kits with spin column bindings.

The purity and integrity of the isolated RNA is a vital step in RNA dependent assays. Here, we present a practical, rapid, and inexpensive method to extract RNA from a small quantity of undamaged pancreatic tissue.

Extraction rapide et rentable de l’ARN du tissu pancréatique rat

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

Biochimie

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Analyse transcriptomique unicellulaire de cellules endocrines pancréatiques de souris

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

Souris îlot de Langerhans isolement à l&#39;aide d&#39;une combinaison de la collagénase purifiée et protéase neutre

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Une méthode pour la souris Isolement îlots pancréatiques et AMPc intracellulaire Détermination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

Isolement et culture de cellules primaires de souris acineuses du pancréas

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Isolement de l'ARN de souris Pancréas: un tissu de Ribonuclease riche

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