Name: rna isolation from mouse pancreas: a ribonuclease-rich tissue
Uploaded: 2014-08-02T14:00:00.000+00:00
Duration: 8 min 47 s
Description: Watch this Scientific Journal Video about RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue at JoVE.com

Ringraziamo Jianhua Ling, Raymond MacDonald, Galvin Swift, Michelle Griffin, Paolo Grippo e Satyanarayana Rachagani per i loro utili commenti, suggerimenti e condivisione delle loro protocolli. Questo lavoro è stato sostenuto da U01CA111294 sovvenzione e un premio di sviluppo dell&#39;idea del programma di ricerca intramurale Ohio State University.

1. Preparazione Le seguenti pratiche aderiscono ai criteri impostati da cura degli animali ed uso commissione dell&#39;Istituzione (IACUC). <ol><li> Si raccomanda di isolare non più di 6 pancreas di topo in un dato giorno. Hanno tutti gli strumenti chirurgici puliti e sterilizzati in autoclave, un set per ogni animale. </li><li> Etichettare tutti i tubi microcentrifuga. Quattro 2 ml provette per animali. </li><li> Mettere 8 ml di reagente di lisi in 50 ml provette da centrifuga su ghiaccio. Nota: Mentre il kit di purificazione viene fornito con un guanidina tiocianato reagente fenolo che è adatto per l&#39;isolamento pancreas, reagente di lisi viene utilizzato a causa dei grandi volumi che sono necessari per l&#39;isolamento. </li><li> Versare circa 10 ml delle seguenti soluzioni in provette da 15 ml per centrifuga per la pulizia di spazzole del omogeneizzatore. Etanolo, 70% (2 tubi), acqua di qualità per HPLC (1 tubo) e acqua di qualità per HPLC contenente circa 200 ml di RNase via o inattivatore ribonucleasi equivalente. </li></ol&gt; 2. Chirurgia, Pancreas Dissection e omogeneizzazione <ol><li> Mettere 1-2 ml di isoflurano in un barattolo contenente garza di cotone o tovaglioli di carta. I topi sono posti su una piattaforma che impedisce loro di raggiungere la fonte del isoflurano. Aggiungi isoflurano fresco al vaso prima di anestetizzare ogni mouse. </li><li> Posizionare delicatamente il mouse nel barattolo contenente il isoflurano. Tappare il vaso e monitorare gli effetti dell&#39;anestesia ruotando delicatamente il barattolo avanti e indietro. L&#39;animale viene anestetizzato una volta che è in grado di stare nel suo &#39;. </li><li> Per la conferma di anestesia, togliere l&#39;animale dal vaso e applicare un pizzico punta ditta. Se il mouse non reagisce al pizzico punta, passare al punto 2.5. </li><li> Se il mouse reagisce pizzico punta, quindi ripetere i passaggi 2,2-2,3. </li><li> Posizionare l&#39;animale anestetizzato sul banco in posizione prona. Dislocare cervice del mouse ermeticamente ponendo la mano sinistra sul collo del mouse. Noizione la mano destra afferrare la coda del topo e tirare verso l&#39;alto ad un angolo di circa 45 ° per dislocare la cervice rapidamente. </li><li> Fissare la carcassa di una superficie (un pezzo livello di polistirolo ricoperto di carta assorbente) da piercing ciascuno dei quattro zampe dell&#39;animale nella superficie piatta usando 25 calibro aghi ipodermici. </li><li> Utilizzando strumenti chirurgici sterili, tagliare la parte mediana del mouse aperta e rapidamente rimuovere il pancreas dal mouse. Utilizzare le pinze per tenere il pancreas e tagliarlo dalla milza e intestino tenue con le forbici a molla. Fare attenzione a non allungare il pancreas durante la cancellazione dal mouse. Turbativa del tessuto potrebbe rilasciare ribonucleasi. Nota: Il tempo necessario per rimuovere il pancreas dal mouse è critica. Un esperto dovrebbe essere in grado di sezionare pancreas di circa 1 minuto o meno. </li><li> Porre l&#39;intero pancreas in una provetta da 50 ml contenente 8 ml di ghiaccio freddo reagente di lisi. Tappare la provetta e brevemente agitare per immergere il tiSSUE nel reagente e procedere alla fase successiva senza indugio. Il rapporto di reagente di lisi di tessuto è critica. Vedere la sezione Risultati per un confronto dell&#39;integrità dell&#39;RNA isolato utilizzando diversi volumi di reagente di lisi. </li><li> Omogeneizzare per 5 sec. È importante premere le lame omogeneizzatore al fondo della provetta da centrifuga per permettere al tessuto di essere tirato nelle lame di omogeneizzazione efficiente. </li></ol> 3. Rimozione di tessuto non digerito Nota: Dopo omogeneizzazione, piccoli frammenti di tessuto rimarranno nel reagente di lisi. E &#39;più importante per la lisi rapidamente il tessuto lasciando un po&#39; di tessuto non digerito, piuttosto che su di omogeneizzazione e il degrado rischio di RNA. <ol><li> Trasferimento di 2 ml di reagente di lisi contenente il pancreas lisati di una provetta 2 ml. Raccogliere due tubi microcentrifuga di omogenato per isolamento. Eliminare l&#39;omogeneizzato rimanente o conservare a -80 ° C per gli studi futuri. </li><li> Centrifuge a 4 ° C, 12000 × g per 5 min a pellet tessuto non digerito. Questo è un passo fondamentale, come riporto di tessuto non digerito nelle successive soluzioni di RNA probabilmente contaminare il campione con ribonucleasi (punto 5.13). </li><li> Trasferire 1 ml del supernatante in una provetta 2 ml. Procedere con l&#39;isolamento subito e memorizzare il tubo replica a -80 ° C per un uso futuro. Posizionare tutte le provette contenenti omogeneizzato sul ghiaccio bagnato mentre si procede alla fase successiva. </li><li> Smaltire il restante reagente di lisi in un apposito contenitore di rifiuti chimici. </li></ol> 4. Pulizia delle Blades Omogeneizzatore <ol><li> Prima di procedere all&#39;isolamento successivo, pulire le lame omogeneizzatore mettendoli in 10 ml di etanolo al 70%. Attivare l&#39;omogeneizzatore per circa 20 sec per rimuovere eventuali pezzi di tessuto che può essere catturato nelle lame. </li><li> Utilizzare una punta di 200 microlitri pipetta per rimuovere eventuali pezzi che rimangono nelle lame omogeneizzatore. </li&gt; <li> Ripetere il punto 4.1 in acqua, generale inattivatore Rnase e il 70% di etanolo. Asciugare l&#39;albero omogeneizzatore con Kim pulito pulire. </li><li> Ripetere il punto 2.1 per l&#39;isolamento pancreas dei rimanenti animali, mantenendo RNA isolato da ogni animale separato piuttosto che mettere in comune l&#39;RNA. </li></ol> 5. Isolamento RNA La sezione seguente è identico al protocollo del kit di purificazione. Il vantaggio del kit di purificazione è che sarà isolare l&#39;RNA totale compreso piccoli RNA. <ol><li> Se si utilizza la soluzione omogeneizzato congelato, scongelare il ghiaccio umido e lasciate riposare a temperatura ambiente per 5 min. </li><li> Aggiungere 200 ml di cloroformio, tappare la provetta e agitare vigorosamente a mano per 15 sec. Lasciate che il tubo di riposare a temperatura ambiente per 2-3 min. </li><li> Centrifugare per 15 minuti a 12.000 xg, a 4 ° C. </li><li> Trasferire lo strato acquoso superiore in una provetta. Aggiungere 1,5 volumi di isopropanolo e mescolare accuratamente pipettando su e giù parecchie volte. </li><li> Trasferimento fino a 700 microlitri di campione in una colonna di spin che viene inserito in un tubo di raccolta da 2 ml. Chiudere il coperchio e centrifugare a 8000 xg per 15 secondi a temperatura ambiente. </li><li> Gettare il flow-through. </li><li> Ripetere i passaggi 5,5-5,6, con il resto del campione. </li><li> Aggiungere 700 microlitri Buffer RWT alla colonna di spin. Centrifugare per 15 sec a 8.000 x g. Gettare il flow-through. </li><li> Dispensare 500 microlitri Buffer RPE nella colonna di spin. Centrifugare per 15 sec a 8000 xg per lavare la colonna. Gettare il flow-through. </li><li> Aggiungere 500 microlitri Buffer RPE alla colonna di spin. Centrifugare per 2 minuti a 8000 xg per asciugare la colonna. </li><li> Trasferire la colonna di selezione per una provetta da 1,5 ml. Dispensare 80 microlitri acqua priva di ribonucleasi direttamente sulla membrana colonna di spin. Centrifugare per 1 min a 8000 xg per eluire l&#39;RNA. </li><li> Aggiungere inibitore RNasi 1 microlitri di soluzione di RNA. Conservare a -80 ° C o procedere alla 5.13. </li><li> Per convalidare l&#39;integrità dell&#39;RNA, prima calcolare il conce RNAntration e 260/280 rapporto utilizzando uno spettrofotometro. </li><li> Diluire una porzione della soluzione di RNA a circa 500 ng / ml con acqua per biologia molecolare. </li><li> Determinare l&#39;RNA Integrity (cioè, RIN) utilizzando l&#39;analisi di elettroforesi capillare. Circa 5 ml di soluzione di RNA è sufficiente per l&#39;analisi. </li><li> Aliquotare la soluzione RNA (vedere Figura 2) e conservare a -80 ° C. </li></ol>

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	<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.

Isolare RNA da tessuti che sono ribonucleasi ricco rappresenta una grande sfida per esperimenti di biologia molecolare. I vari reagenti e kit sono disponibili in commercio che si basano principalmente sul metodo fenolo guanidina tiocianato di estrazione di RNA. Guanidina tiocianato denatura proteine ​​e quindi riduce l&#39;attività di ribonucleasi. Tuttavia, l&#39;ampiezza delle ribonucleasi nel pancreas richiede ulteriori modifiche ai protocolli standard di isolamento dell&#39;RNA. Un protocollo è riportato qui c...

È necessario isolamento di RNA con grande integrità per esperimenti di routine di biologia molecolare come Northern blotting 9, qRT-PCR 1 o gene expression profiling 5. La maggior parte dei metodi contemporanei di isolamento dell&#39;RNA si basano sulle modificazioni dei protocolli tiocianato di guanidina 2, 3. Guanidina tiocianato è un forte proteina denaturante e verrà efficacemente interrompere l&#39;attività di ribonucleasi in molte condizioni. Il metodo popolare di Chomczynski e Sacchi 3 combinato fenolo alla soluzione di guanidina tiocianato lisi, riducendo il tempo di isolamento per circa 4 ore. Molti reagenti di estrazione dell&#39;RNA commercialmente disponibili sono basati sul metodo Chomczynski e Sacchi 3. Ribonucleasi ricchi tessuti come umano o il mouse pancreas presenta una sfida aggiuntiva per isolare RNA. Pancreas mouse può contenere fino a 75 mg di ribonucleasi 6, alcune delle quali verrà rilasciato During rottura del tessuto pancreatico conseguente autolisi tessuto. Modifiche dei protocolli guanidina tiocianato sono stati utilizzati con successo per isolare l&#39;RNA da pancreas con alta integrità 2, 4, 7, 10. L&#39;infusione di RNA stabilizzante in topo pancreas facilitato isolamento di RNA con elevata integrità 4, 7, 10, tuttavia infusione di Le soluzioni nel pancreas richiede grande abilità, possono richiedere strumenti specializzati come un microscopio da dissezione e interrompere architettura del tessuto durante la procedura può provocare la lisi cellulare. Perfusione tissutale con reagente stabilizzazione dell&#39;RNA potrebbe interferire con altre applicazioni, tra cui l&#39;isolamento delle proteine ​​e la colorazione istologica. Inoltre, questa tecnica non è adatto per isolare l&#39;RNA da pancreas umano. Altri protocolli richiedono la preparazione di soluzioni specifiche dallo sperimentatore 2. Riportiamo un protocollo per isolare l&#39;RNA con grande integrità di topo pancreas. The protocollo utilizza elementi dei metodi precedentemente pubblicati ed è in gran parte basata su protocolli standard di fenolo / guanidina a base di tiocianato-lisi reagenti. Non richiede la preparazione di soluzioni specializzate né richiede infusione di reagenti nelle pancreas. Passaggi critici per il successo di isolamento di RNA comprendono un elevato fenolo / guanidina-basato lisi reagente rapporto tra tessuto, la rimozione del tessuto non digerito prima fase la separazione e l&#39;inclusione di un inibitore della ribonucleasi alla soluzione RNA risultante. Utilizzando queste e altre modifiche, di solito isolare l&#39;RNA con RIN superiore a 7 da utilizzare per l&#39;analisi di espressione genica di routine.

<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.

La resa RNA totale da 1 ml di omogenato lisi è 20-40 mg. OD 260/280 rapporti sono in genere circa il 2,0 e il RIN sono costantemente maggiore di 7.0. Se il RIN è ≤ 6, l&#39;isolamento dovrà essere ripetuta. Occasionalmente, un RIN che è superiore a 8,0 si ottiene. I due metodi più comunemente usati di eutanasia topi prima della rimozione del pancreas sono CO 2 asfissia o inalazione di isoflurano. Entrambe le tecniche sono seguiti da dislocazione cervicale. Poiché è possib...

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.

Isolamento dell&#39;RNA da mouse Pancreas: Un Tessuto ricco Ribonucleasi

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

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

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.

Murino pancreatica isolamento Islet

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Biologia

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 saggi per Caratterizzazione Tri-potenti cellule progenitrici isolato dal adulti murini 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. ...

Biologia dello sviluppo

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.

Efficace isolamento di isole funzionali da neonatali mouse Pancreas

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.

Sequenziamento dell'RNA a singola cellula e analisi delle isole pancreatiche umane

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

Genetica

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.

Estrazione rapida ed economica dell'RNA del tessuto pancreatico ratto

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

Biochimica

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.

Analisi trascrittomica unicellulare delle cellule Endocrine del pancreas del Mouse

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.

Mouse Isolotto di Langerhans isolamento utilizzando una combinazione di Collagenasi depurato e proteasi neutra

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 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.

Un semplice protocollo ad alta efficienza per l'isolamento delle isolozioni pancreatiche dai topi

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Profiling dell'espressione genica specifica del tipo di cella nel fegato del mouse

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

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.

Isolamento dell'RNA da mouse Pancreas: Un Tessuto ricco Ribonucleasi

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