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

우리는 그들의 도움이 의견, 제안 및 자신의 프로토콜을 공유 지안 후아 링, 레이몬드 맥도날드, 갤빈 스위프트, 미셸 그리핀, 폴 Grippo 및 Satyanarayana Rachagani 감사합니다. 이 작품은 부여 U01CA111294와 오하이오 주립 대학 교내 연구 프로그램에서 아이디어 개발의 수상에 의해 지원되었다.

1. 준비 다음 사례는 기관의 동물 관리 및 사용위원회 (IACUC)에 의해 설정된 정책을 준수. <ol><li> 그것은 특정 일에 더 이상 6 이상 마우스 췌장을 분리하는 것이 좋습니다. 깨끗하고 멸균 모든 수술 도구, 동물 당 하나의 집합이 있습니다. </li><li> 모든 마이크로 원심 튜브 레이블을 지정합니다. 동물 당 네 개의 2 ㎖ 튜브. </li><li> 얼음에 50 ㎖의 원심 분리기 튜브에 용해 시약 8 ㎖를 놓습니다. 주 : 정제 키트는 췌장의 분리에 적합한 구아니딘 티오 시아 네이트 페놀 시약와 함께 제공하지만, 용해 시약 때문에 격리 당 필요한 큰 볼륨으로 사용됩니다. </li><li> 균질의 블레이드 청소를 위해 15 ML의 원심 분리기 튜브에 다음과 같은 솔루션의 약 10 ㎖를 분배. 에탄올, 70 % (2 관), HPLC 등급 물 (1 관)과 약 200의 RNase ㎕의 비움 또는 이에 상응하는 리보 뉴 클레아 제 inactivator를 포함 HPLC 등급 물. </li></oL&gt; 2. 외과, 췌장의 해부 및 균질화 <ol><li> 면 거즈 나 종이 수건이 들어있는 항아리에 이소 플루 란의 1 ~ 2 ㎖를 놓습니다. 마우스는 이소 플루 란의 근원에 도달 할 수는없는 플랫폼에 배치됩니다. 이전에 각 마우스를 마취에 항아리에 신선한 이소 플루 란을 추가합니다. </li><li> 부드럽게 이소 플루 란을 포함하는 항아리에 마우스를 놓습니다. 항아리 뚜껑을 부드럽게 앞뒤로 항아리를 회전하여 마취의 효과를 모니터링 할 수 있습니다. 그것의 자신에 서 할 수 없습니다에 일단 동물은 마취. </li><li> 마취의 확인을 위해, 항아리에서 동물을 제거하고 회사 발가락 핀치를 적용합니다. 마우스가 발가락 핀치에 반응하지 않는 경우, 2.5 단계로 진행합니다. </li><li> 마우스가 발가락 핀치에 반응하는 경우, 다음 단계 2.3에 2.2를 반복합니다. </li><li> 발생하기 쉬운 위치에 벤치 위에 마취 동물을 놓습니다. 단단히 쥐의 자궁 경부의 왼쪽 손을 배치하여 마우스의 자궁 경부 탈구. 우리오른쪽 마우스의 꼬리를 잡고 빨리 자궁 경부 탈구에 약 위쪽으로 45 °의 각도를 끌어 보내고. </li><li> 25 게이지 피하 주사 바늘을 사용하여 평평한 표면에 동물의 네 발을 각각 관통하여 표면 (종이 타월로 덮여 스티로폼 수준의 조각)에 시체를 고정합니다. </li><li> 오픈 마우스의 중간 부분을 절단하고 신속하게 마우스의 췌장을 제거, 무균 수술 도구를 사용하여. 췌장을 보유하고 봄 가위를 사용하여 비장과 소장에서 잘라 집게를 사용합니다. 마우스에서 제거하는 동안 췌장을 스트레칭하지 않도록주의하십시오. 조직의 혼란은 리보 뉴 클레아 제를 해제 할 수 있습니다. 참고 : 마우스의 췌장을 제거하는 데 걸리는 시간이 중요합니다. 당업자는 약 1 분 이하로 췌장 해부를 할 수 있어야한다. </li><li> 차가운 얼음 용해 시약 8 ㎖를 포함하는 50 ML 튜브로 전체 췌장을 놓습니다. 튜브 캡을 간략하게 TI 젖어 흔들시약에 ssue 지체없이 다음 단계로 진행합니다. 조직에 용균 시약의 비율이 중요하다. 용해 시약의 다른 볼륨을 사용하여 격리 RNA 무결성의 비교 결과 섹션을 참조하십시오. </li><li> 5 초 동안 균질화한다. 그것은 조직이 효율적 균질화 블레이드로 당겨질 수 있도록 원심 관의 바닥에 균질 블레이드를 눌러야 중요하다. </li></ol> 소화되지 않은 조직의 3. 제거 참고 : 균질화에 따라, 조직의 작은 비트는 용해 시약에 남아 있습니다. 그것은 신속하게 균질화와 RNA의 위험 저하에보다는 소화되지 않은 일부 조직을 떠나는 조직을 용해하는 것이 더 중요합니다. <ol><li> 전송 2 ㎖ 마이크로 원심 튜브에 용해 췌장을 포함하는 세포 용해 시약 2 ㎖. 격리 당 균질의 두 마이크로 원심 튜브를 수집합니다. 미래 연구를위한 -80 ° C에서 나머지 균질 또는 상점을 폐기하십시오. </li><li> 센트4 ° C, 소화되지 않은 조직을 펠렛 5 분간 12,000 × g에서 rifuge. 이 가능성이 리보 뉴 클레아 제 (단계 5.13)로 샘플을 오염시킬 것 RNA의 후속 솔루션으로 소화되지 않은 조직의 이월 등의 중요한 단계입니다. </li><li> 2 ㎖ 마이크로 원심 분리 튜브에 이송 상등액 1 ㎖. 즉시 격리을 진행하고 향후 사용을 위해 -80 ° C에서 복제 튜브를 저장합니다. 다음 단계를 진행하는 동안 얼음물에 균질를 포함하는 모든 튜브를 놓습니다. </li><li> 적절한 화학 폐기물 용기에 남아있는 용해 시약을 폐기하십시오. </li></ol> 균질 블레이드 4. 청소 <ol><li> 다음 분리를 진행하기 전에, 70 % 에탄올 10ml에 넣어서 균질화 블레이드 청소. 블레이드에 잡힐 수 있습니다 조직의 덩어리를 제거하기 위해 20 초 동안 약의 균질화를 활성화합니다. </li><li> 균질 블레이드에 남아있는 조각을 제거하기 위해 200 μL 피펫 팁을 사용합니다. </lI&gt; <li> 물, 일반의 RNase inactivator 및 70 % 에탄올의 단계를 반복합니다 4.1. 깨끗한 김 균질 샤프트 닦아 건조. </li><li> 나머지 동물의 췌장 분리 단계를 반복 2.1, 오히려 RNA를 풀링보다 각 동물 별도의 분리 유지 RNA. </li></ol> 5. RNA 격리 다음 섹션에서는 정제 키트 프로토콜과 동일하다. 정제 키트의 장점은 작은 RNA를 포함하여 전체 RNA를 분리한다는 것이다. <ol><li> 냉동 균질 솔루션을 사용하는 경우, 5 분 동안 실온에서 스탠드를 얼음물에 해동 및하자. </li><li> 클로로포름 200 μl를 추가, 튜브 캡을 15 초 동안 손으로 격렬하게 흔들어. 튜브 2 ~ 3 분 동안 RT에 서 보자. </li><li> 12,000 XG, 4 ℃에서 15 분 동안 원심 분리 </li><li> microcentrifuge 관에 위 수성 레이어를 전송합니다. 이소프로판올 1.5 볼륨을 추가로 pipetting 아래로 여러 번 철저하게 섞는다. </li><li> 70까지 이동2 ML 수집 관에 위치 스핀 컬럼에 샘플의 0 μL. 실온에서 15 초 동안 8,000 XG에 원심 분리기 뚜껑을 닫습니다. </li><li> 흐름을 통해 폐기하십시오. </li><li> 반복 샘플의 나머지 부분을 사용하여, 5.5-5.6 단계를 반복합니다. </li><li> 스핀 컬럼에 700 μL 버퍼 RWT를 추가합니다. 8,000 X g에서 15 초 동안 원심 분리기. 흐름을 통해 폐기하십시오. </li><li> 스핀 칼럼에 피펫 500 μL 버퍼 RPE. 열을 씻어 8,000 XG에 15 초 동안 원심 분리기. 흐름을 통해 폐기하십시오. </li><li> 스핀 컬럼에 500 μL 버퍼 RPE를 추가합니다. 열을 건조하는 8,000 XG에서 2 분 동안 원심 분리기. </li><li> 1.5 ML의 microcentrifuge 관에 스핀 열을 전송합니다. 직접 스핀 컬럼 막 상 피펫 80 μL의 리보 뉴 클레아없는 물. RNA를 용출 8,000 XG에서 1 분 동안 원심 분리기. </li><li> RNA 용액 1 μL의 RNA 분해 효소 억제제를 추가합니다. -80 ° C에서 저장 또는 5.13로 진행합니다. </li><li> RNA의 무결성을 확인하려면 먼저 RNA의 conce을 계산분광 광도계를 사용하고 ntration 280분의 260 비. </li><li> 분자 생물학 수준의 물에 약 500 NG / μL로 RNA 용액의 일부를 희석. </li><li> 모세관 전기 영동 분석을 사용하여 RNA 무결성 (즉, RIN)를 확인합니다. RNA 용액을 약 5 μL는 분석에 충분하다. </li><li> C. -80 ° RNA 용액 (그림 2 참조) 및 저장소를 나누어지는 </li></ol>

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The authors have nothing to disclose.

풍부한 리보 뉴 클레아하는 조직에서 RNA를 분리하는 것은 분자 생물학 실험에 대한 큰 도전을 나타냅니다. 각종 시약 및 키트는 주로 RNA 추출의 페놀 구아니딘 티오 시아 네이트 방법에 시판을 기반으로하는 그. 구아니딘 티오 시아 네이트는 단백질을 변성 따라서 리보 뉴 클레아 제의 활성을 감소시킨다. 그러나 췌장에서 리보 뉴 클레아 제의 깎아 지른듯한 크기는 RNA 분리의 표준 프로토콜을 추...

높은 무결성 RNA의 분리는 같은 북부 블로 팅 9, QRT-PCR 1 또는 5 프로파일 유전자 발현 같은 일상적인 분자 생물학 실험이 필요합니다. RNA 분리 대부분의 현대적인 방법을 구아니딘 티오 시아 네이트 프로토콜 (2)의 변형에 기초한다 3. 구아니딘 티오 시아 네이트는 강한 단백질 변성제이고 효과적으로 대부분의 조건에서 리보 뉴 클레아 제의 활성을 방해 할 것이다. Chomczynski 및 사키 3의 인기있는 방법은 약 4 시간에 분리 시간을 줄이고, 구아니딘 티오 시아 네이트 용해 용액에 페놀을 배합. 대부분의 시판되는 RNA 추출 시약은 Chomczynski와 사키 방법 3를 기반으로하고 있습니다. 같은 인간이나 마우스의 췌장 등의 리보 뉴 클레아 풍부한 조직은 RNA를 분리하는 추가 문제를 제공합니다. 마우스의 췌장은 그 중 일부는 운영 시간에만 출시 될 예정 리보 뉴 클레아 제 6의 최대 75 밀리그램을 포함 할 수있다G 조직의자가 분해의 결과로 췌장 조직의 파괴. 구아니딘 티오 시아 네이트 프로토콜의 수정은 성공적으로 높은 무결성 2, 4, 7, 10 높은 무결성 4, 7, 10와 RNA의 마우스 췌장 용이 분리로 RNA 안정화 시약. 주입 그러나 주입으로 췌장으로부터 RNA를 분리하는 데 사용되어왔다 췌장에 솔루션은 좋은 기술을 필요로 같은 해부 현미경 및 절차를 수행하는 동안 조직 구조를 방해하는 세포 용해 될 수 있습니다와 같은 전문 장비가 필요할 수 있습니다. RNA 안정화 시약과 조직을 관류하는 단백질 분리 및 조직 학적 염색 등의 다른 응용 프로그램을 방해 할 수 있습니다. 또한,이 기술은 인간의 췌장에서 RNA를 분리에는 적합하지 않다. 다른 프로토콜은 조사 (2)에 의해 특정 솔루션의 준비가 필요합니다. 우리는 마우스의 췌장에서 높은 무결성과 RNA를 분리하는 프로토콜을보고합니다. 일E 프로토콜은 이전에 발행 된 방법의 요소들을 사용하여 주로 표준 페놀 / 구아니딘 티오 시아 네이트 계 용해 시약 프로토콜에 기초한다. 이것은 특수한 용액의 제조를 필요로하지 않으며 이는 췌장에 시약의 주입을 요구한다. 성공적인 RNA 격리를위한 중요한 단계는 조직의 비율, 그 결과 RNA 용액에 분리 리보 뉴 클레아 제 억제제의 포함을 단계적으로 이전에 소화되지 않은 조직의 제거에 높은 페놀 / 구아니딘 계 용해 시약 있습니다. 이들 및 다른 변형을 사용하여, 우리는 일반적으로 12보다 큰 루틴 유전자 발현 해석에 사용하는 RIN으로 RNA를 분리.

<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.

용해 균질 1 ㎖에서 총 RNA의 수율은 20 ~ 40 μg이다. OD 280분의 260 비율은 일반적으로 약 2.0이며, RIN은 7.0 지속적으로 크다. RIN은 6 ≤ 경우 분리가 반복 될 필요가있을 것이다. 때때로, 8.0보다 높은 R​​IN은 달성된다. 종래 췌장의 제거에 마우스를 안락사의이 가장 일반적으로 사용되는 방법은 CO 2 질식 또는 이소 플루 란을 흡입한다. 두 기술은 자궁 경부 전위 다음에 ?...

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.

마우스 췌장에서 RNA 격리 : 리보 뉴 클레아 풍부한 조직

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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JoVE Journal

Biology

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

비디오: 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.

Murine 췌장 아일릿 격리

연구

생물학

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.

체외 식민지 분석 실험에서 트라이 강력한 전구 세포를 특성화를위한 성인 쥐 췌장에서 분리

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. ...

발생학

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.

신생아 마우스 췌장의 기능 독도의 효과적인 분리

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.

단세포 RNA 시퀀싱 및 인간 췌장 섬 분석

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

유전학

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.

쥐 췌장 조직의 신속하고 비용 효율적인 RNA 추출

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

생화학

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.

마우스 췌 장 내 분 비 세포의 단일 셀 Transcriptomic 분석

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.

Langerhans 절연의 마우스 작은 섬은 정화의 Collagenase와 중성 단백 분해 효소의 조합을 사용하여

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.

마우스에서 췌장 췌도 격리를 위한 간단한 고효율 프로토콜

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.

마우스 간에서 세포 유형 별 유전자 발현 프로파일링

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.