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

Agradecemos Jianhua Ling, Raymond MacDonald, Galvin Swift, Michelle Griffin, Paul Grippo e Satyanarayana Rachagani por seus comentários úteis, sugestões e partilha de seus protocolos. Este trabalho foi apoiado por U01CA111294 concessão e um Prêmio Idea Desenvolvimento do Programa de Pesquisa intramuros Ohio State University.

1. Preparação As seguintes práticas aderir às políticas estabelecidas pelo Animal Care and Use Committee da Instituição (IACUC). <ol><li> Recomenda-se a isolar não mais de 6 pâncreas do rato em qualquer dia. Tenha todos os instrumentos cirúrgicos limpos e autoclavados, um conjunto por animal. </li><li> Rotular todos os microtubos. Quatro tubos de 2 ml por animal. </li><li> Colocar 8 ml de reagente de lise em 50 ml de tubos de centrífuga em gelo. Nota: Embora o kit de purificação trata com um reagente fenol de tiocianato de guanidina, que é adequado para o isolamento de pâncreas, de reagente de lise é usado por causa dos grandes volumes de que são necessárias por isolamento. </li><li> Dispensar cerca de 10 ml das seguintes soluções em tubos de centrífuga de 15 ml para a limpeza lâminas do homogeneizador. O etanol, 70% (2 tubos), HPLC água (1 tubo) e água de grau HPLC, contendo cerca de 200 mL de RNase afastado ou inativador ribonuclease equivalente. </li></ol&gt; 2. Cirurgia, Dissection Pâncreas e homogeneização <ol><li> Coloque 1-2 ml de isoflurano em um frasco contendo gaze de algodão ou toalhas de papel. Os ratos são colocados numa plataforma que os impede de se atingir a fonte de isoflurano. Adicionar isoflurano fresco para o frasco antes de anestesiar cada rato. </li><li> Delicadamente, coloque o mouse no frasco contendo o isoflurano. Tampe o frasco e monitorar os efeitos da anestesia, rodando suavemente o frasco e para trás. O animal é anestesiado, uma vez que está na incapaz de ficar na sua &quot;própria. </li><li> Para confirmação da anestesia, remover o animal do frasco e aplicar uma pitada do dedo do pé firme. Se o mouse não reagir ao aperto do dedo do pé, vá para o passo 2.5. </li><li> Se o rato reage ao pitada dedo do pé, em seguida, repita os passos de 2,2-2,3. </li><li> Colocar o animal anestesiado na bancada na posição prona. Deslocar colo do rato por força colocando a mão esquerda no colo do mouse. Nósção da mão direita agarrar a cauda do rato e rapidamente puxar para cima em um ângulo de aproximadamente 45 ° para deslocar o colo do útero. </li><li> Fixe a carcaça a uma superfície (um pedaço de isopor nível de cobertas com toalhas de papel), perfurando cada uma das quatro patas do animal na superfície plana usando calibre 25 agulhas hipodérmicas. </li><li> Usando instrumentos cirúrgicos estéreis, corte o meio do mouse aberta e rapidamente remover o pâncreas do mouse. Use a pinça para segurar o pâncreas e cortá-la a partir do baço e do intestino delgado com uma tesoura de mola. Tenha cuidado para não esticar o pâncreas durante a remoção do mouse. O rompimento do tecido poderia liberar ribonuclease. Nota: O tempo que leva a remover o pâncreas do rato é crítica. Um perito deve ser capaz de dissecar o pâncreas em cerca de um minuto ou menos. </li><li> Colocar todo o pâncreas para um tubo de 50 ml contendo 8 ml de reagente de lise frio de gelo. Tampe o tubo e agitar brevemente para imergir o tissue no reagente e avançar para o próximo passo sem demora. A proporção de reagente de lise de tecido é crítica. Ver secção de resultados de uma comparação entre a integridade do RNA isolado utilizando diferentes volumes de reagente de lise. </li><li> Homogeneizar por 5 seg. É importante para pressionar as lâminas homogeneizador para o fundo do tubo de centrífuga para permitir que o tecido seja puxado para dentro das lâminas para homogenização eficiente. </li></ol> 3. Remoção de tecido não digerido Nota: Após a homogeneização, pequenos pedaços de tecido permanecem no reagente de lise. É mais importante para lisar rapidamente o tecido deixando um pouco de tecido não digerido, em vez de ao longo de homogeneização e degradação risco de RNA. <ol><li> Transferência de 2 ml de reagente de lise contendo o pâncreas lisadas para um tubo de microcentrífuga de 2 ml. Colete dois microtubos de homogeneizado por isolamento. Descarte o homogeneizado restante ou armazenar a -80 ° C para estudos futuros. </li><li> Centavorifuge a 4 ° C, 12000 g durante 5 min para sedimentar o tecido não digerido. Este é um passo crítico como transição de tecido não digerido em soluções posteriores de RNA provavelmente vai contaminar a amostra com ribonuclease (passo 5.13). </li><li> Transferir 1 ml do sobrenadante para um tubo de microcentrifugação de 2 ml. Prosseguir com o isolamento imediatamente e armazenar o tubo de replicar-se a -80 ° C para uso futuro. Coloque todos os tubos contendo homogeneizado em gelo molhado enquanto prosseguir para a próxima etapa. </li><li> Descarte o reagente de lise restante em um recipiente de lixo químico apropriado. </li></ol> 4. Limpeza dos Blades homogeneizador <ol><li> Antes de prosseguir para o próximo isolamento, limpeza das lâminas homogeneizador, colocando-os em 10 ml de etanol a 70%. Ative o homogeneizador por cerca de 20 segundos para remover quaisquer pedaços de tecido que podem ser capturados nas lâminas. </li><li> Use uma ponta de 200 mL da pipeta para remover quaisquer peças que permanecem nas lâminas homogeneizador. </li&gt; <li> Repita o passo 4.1 na água, inativador geral Rnase e 70% de etanol. Seque o eixo com um homogeneizador de Kim limpo limpar. </li><li> Repetir o passo 2.1 para o isolamento pâncreas dos animais restantes, mantendo RNA isolado a partir de cada animal, em vez de separar o pool de RNA. </li></ol> 5. Isolamento de ARN A secção que se segue é idêntico ao protocolo do kit de purificação. A vantagem do kit de purificação é que vai isolar o ARN total, incluindo pequenos RNAs. <ol><li> Se estiver usando solução homogeneizado congelado, descongelar no gelo molhado e deixe descansar em temperatura ambiente por 5 min. </li><li> Adicionar 200 mL de clorofórmio, tampar o tubo e agitar vigorosamente à mão por 15 segundos. Deixe o tubo repousar à temperatura ambiente durante 2-3 minutos. </li><li> Centrifugar durante 15 minutos a 12.000 xg, a 4 ° C. </li><li> Transferir a fase aquosa superior para um tubo de microcentrífuga. Adicionar 1,5 volumes de isopropanol e misture bem por pipetagem cima e para baixo várias vezes. </li><li> Transfira até 700 ul da amostra em uma coluna de rotação que é colocada num tubo de recolha de 2 ml. Fechar a tampa e centrifugar a 8000 g durante 15 segundos à temperatura ambiente. </li><li> Descartar o escoamento. </li><li> Repita os passos de 5,5-5,6, utilizando-se o restante da amostra. </li><li> Adicionar 700 mL de buffer ERP para a coluna de centrifugação. Centrifugar durante 15 segundos a 8.000 x g. Descartar o escoamento. </li><li> Pipetar 500 mL de buffer RPE na coluna de rotação. Centrifugar durante 15 segundos a 8000 x g para lavar a coluna. Descartar o escoamento. </li><li> Adicionar 500 mL de buffer RPE para a coluna de centrifugação. Centrifugar durante 2 minutos a 8.000 xg, para secar a coluna. </li><li> Transferir a coluna de centrifugação para um tubo de microcentrífuga de 1,5 ml. Pipetar 80 mL sem ribonuclease água diretamente sobre a membrana coluna rodada. Centrifugar durante 1 minuto a 8.000 xg, para eluir o ARN. </li><li> Adicionar 1 mL inibidor de RNase à solução de ARN. Armazenar a -80 ° C ou prosseguir para 5.13. </li><li> Para validar a integridade do RNA, primeiro calcular o conce RNAntration e 260/280 escala utilizando um espectrofotômetro. </li><li> Dilui-se uma porção da solução de RNA de cerca de 500 ng / mL com água de grau de biologia molecular. </li><li> Determinar a integridade de ARN (isto é, RIN) usando a análise de electroforese capilar. Cerca de 5 ul da solução de RNA é suficiente para a análise. </li><li> Alíquota da solução de RNA (ver Figura 2) e armazenar 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.

Isolar RNA a partir de tecidos que são ribonuclease rico representa um grande desafio para os experimentos de biologia molecular. Vários reagentes e kits estão disponíveis no mercado que são baseada principalmente no método fenol tiocianato de guanidina de extração de RNA. Tiocianato de guanidina desnatura a proteína e, assim, reduz a actividade da ribonuclease. No entanto, a magnitude da ribonuclease no pâncreas requer modificações adicionais para protocolos padrão de isolamento de RNA. Um protocolo é rel...

Isolamento de RNA com elevada integridade é necessária para experiências de biologia molecular de rotina, tais como transferência Northern 9, qRT-PCR 1 ou a expressão do gene de perfil 5. A maioria dos métodos contemporâneos de isolamento de ARN são baseados em modificações dos protocolos de tiocianato de guanidina 2, 3. Tiocianato de guanidina é uma grande proteína desnaturante e vai efectivamente interromper a actividade de ribonuclease sob a maioria das condições. O método popular de Chomczynski e Sacchi 3 combinada de fenol na solução de tiocianato de guanidina a lise, reduzindo o tempo de isolamento de cerca de 4 horas. Muitos reagentes de extracção de RNA comercialmente disponíveis baseiam-se no método de Chomczynski e Sacchi 3. Ribonuclease tecidos ricos, como pâncreas humano ou mouse apresenta um desafio adicional para isolar RNA. Rato pâncreas pode conter até 75 mg de ribonuclease 6 alguns dos quais será lançado during destruição do tecido pancreático resultante em autólise tecido. As modificações dos protocolos de tiocianato de guanidina foram utilizados com êxito para isolar o ARN a partir de pâncreas com alta integridade 2, 4, 7, 10. Infusão de ARN estabilização reagente em rato pâncreas isolamento facilitada do RNA com elevada integridade 4, 7, 10, no entanto, a infusão de soluções para o pâncreas exige muita destreza, pode exigir instrumentos especializados, como um microscópio de dissecação e interrompendo a arquitetura do tecido durante o procedimento pode resultar em lise celular. Perfusão tecidual com reagente estabilização RNA pode interferir com outras aplicações, incluindo o isolamento de proteínas e coloração histológica. Além disso, esta técnica não é adequada para o isolamento de RNA a partir de pâncreas humanos. Outros protocolos requerem a preparação de soluções específicas pelo pesquisador 2. Relata-se um protocolo para isolar RNA com alta integridade de rato pâncreas. The protocolo utiliza elementos de métodos publicados anteriormente e é em grande parte baseada nos protocolos de reagentes padrão fenol / guanidina à base de tiocianato de lise. Ela não requer a preparação de soluções especializados, nem requer a infusão de reagentes para o pâncreas. As etapas críticas para o isolamento do ARN de sucesso incluem uma alta fenol / à base de guanidina lise reagente à relação do tecido, remoção de tecido não digerido antes da separação de fases e a inclusão de um inibidor da ribonuclease-se à solução de ARN resultantes. Usando estas e outras modificações, que tipicamente isolar o ARN com RIN superior a 7 para ser utilizado para análise da expressão do gene de rotina.

<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.

O rendimento de ARN total a partir de 1 ml de homogeneizado de lise é de 20-40 ug. OD 260/280 proporções são normalmente em torno de 2.0 e RIN são consistentemente superior a 7,0. Se o NIR é ≤ 6, o isolamento necessita de ser repetida. Ocasionalmente, um RIN que é maior do que 8,0 seja alcançado. Os dois métodos mais comumente usados ​​de eutanásia de camundongos antes da remoção do pâncreas são CO 2 asfixia ou inalação de isoflurano. Ambas as técnicas são ...

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 de RNA Rato Pâncreas: Um tecido rico em Ribonuclease

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 ...

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27.5K visualizações.  The Ohio State University. We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Vídeo: 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.

Isolamento Islet murino de pâncreas

Biologia

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.

Isolamento eficaz de ilhotas funcionais a partir de ratinho neonato Pâncreas

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

Biologia do Desenvolvimento

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.

Sequenciamento de RNA de célula única e análise de ilhotas pancreáticas humanas

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

Genética

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

Geração de insulina tridimensional, livre de andaime expressando Pancreatoids do Mouse progenitoras pancreáticas em Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

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.

Extração rápida e econômica do RNA do tecido pancreático de rato

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

Bioquímica

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.

Análises de célula única Transcriptomic de células endócrinas no pâncreas de rato

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.

Rato Ilhéu de Langerhans isolamento usando uma combinação de colagenase purificada e Protease 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 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.

Um Método para Mouse ilhotas pancreáticas Isolamento e intracelular cAMP Determinação

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

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.

Um protocolo de alta eficiência simples para isolamento de Islet pancreático de camundongos

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

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Dinâmica de cálcio de imagem em subpopulações de células de ilhotas pancreáticas do rato

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...