We thank Hongjae Sunwoo for helpful advice for oligonucleotide design in RNA FISH. We also thank Serenity Curtis for editing the manuscript. N.Y. was supported by a Postdoctoral Fellowship for Research Abroad of the Japan Society for the Promotion of Science (JSPS). This work was supported by the NIH (RO1-GM102184), the March of Dimes Research Foundation (#6-FY12-337), and the Developmental Fund and Trustee Grant at Cincinnati Children's Hospital Medical Center to Y.O.

1. 프로브 준비 <ol><li> 5&#39;- 아미노 수정하여 여러 독특한 XIST에 올리고 뉴클레오티드 (20 ~ 30 염기 길이의 올리고 뉴클레오티드, 63 ~ 65 ºC의 용융 온도, 표 1)를 얻어 물을 일시 중지합니다. </li><li> 5&#39; 아미노 수정 (총 4.5 μg의) 제조업체의 지시에 따라 아민 반응성 형광 염료와 레이블 올리고 뉴클레오티드의 풀 몰량. <ol><li> 클레아없는 물 5 μL의 최종 풀링 된 올리고 뉴클레오티드에 용해시키고, 올리고 뉴클레오티드 용액에 1 M 중탄산 나트륨 3 μL를 추가한다. </li><li> 간단히 아민 반응성 염료와 소용돌이의 바이알에 2 μL의 DMSO를 추가합니다. </li><li> DMSO의 반응성 염료와 함께 올리고 뉴클레오티드 용액을 혼합하고 어둠 속에서 실온에서 1 시간 동안 혼합물을 배양한다. </li></ol></li><li> 에탄올 침전 다음에 G-25의 열을 기준으로 형광 표지 된 올리고 뉴클레오티드 프로브를 정화. <ol><li&gt; 750 XG 1 분 원심 분리기 G-25 컬럼은 과잉 저장 버퍼를 제거합니다. </li><li> 단계 1.2.3에서 생산되는 라벨 혼합물에 40 ㎕의 클레아없는 물을 추가하고 G-25 컬럼에 적용됩니다. 2 분 동안 750 XG에 원심 분리기. </li><li> 단계 1.3.2에서 정제 된 프로브로 50 μL 클레아없는 물, 10 ㎕의 3 M 아세트산 나트륨 (PH 5.2) 및 에탄올 250 μL를 추가한다. 4 ° C에서 15 분 동안 21,000 XG에 30 분 원심 분리기에 대한 -80 ° C에서 보관. </li><li> 70 % 에탄올 1 ml로 한 번 침전 올리고 뉴클레오티드를 씻으십시오. </li><li> 50 μL에 재현 탁 클레아없는 물 또는 TE (10 mM 트리스 - 염산, pH가 7.5, 1 mM의 EDTA) 대략 10 μM의 최종 농도. </li></ol></li><li> 혼성화 완충액으로 250 nM의 최종 농도로 형광 표지 된 올리고 뉴클레오티드 프로브를 희석 (10 % 포름 아미드; 2 × SSC [300 mM의 염화나트륨을 30 mM의 시트르산 나트륨], 2 ㎎ / ㎖ BSA, 10 % 덱스 트란 설페이트). 참고 :이 빠른 물고기 프로토콜로, 올리고 뉴클레오티드 페이지XIST 가운에 설계 및 혼성화 배양 시간을 단축하기 위해, 올리고 뉴클레오티드 프로브를 이용하여 FISH 프로토콜 정상보다 더 높은 농도로 사용 하였다. </li></ol> 2. 슬라이드 준비 참고 : 슬라이드가 마우스 배아 줄기 (ES) 또는 배아 체 (EB)를 사용하여 면역 FISH을 준비 할 수 있습니다 폴리 라이신 처리 된 슬라이드에 직접 성장 또는 세포 현탁액과 cytospin를 사용하여 전지 (20), (21)을 차별화. 여기에, cytospin에 의해 슬라이드 준비가 표시됩니다. <ol><li> 6 잘 접시에 문화 매체를 대기음. 철저하게 PBS (실온) 및 흡인 씻으시오. </li><li> 37 ° C에서 5 ~ 10 분 동안 품어, 6 자 접시에 트립신의 0.5 ML을 추가합니다. </li><li> 매체의 5 ML을 추가하고 부드럽게 여러 번을 피펫 아래로 세포를 분산. </li><li> 실온에서 200 XG에서 5 분 동안, 15 ml의 원심 분리 튜브에 세포를 옮긴다. </li><li> 매체를 폐기부드럽게 2 ml의 PBS에 세포를 일시 좋아. </li><li> 혈구 세포를 이용하여 카운트하고, PBS에 의해 4 × 105 세포 / ml에서 세포 현탁액을 희석. </li><li> 슬라이드, 필터 카드와 클립 챔버를 조립하고 cytospin의 회 전자의 해당 슬롯에 배치합니다. cytospin의 조립 단위의 각 실에 단계 2.6에서 제조 된 각 시료의 250 μl를 추가합니다. 실온에서 10 분 동안 1,500 rpm에서 원심 분리기. </li><li> 40 ml의 얼음처럼 차가운 PBS, CSK 버퍼의 각 얼음 3 코 플린 항아리 채우기 (10 mM의 파이프, 산도 6.8, 100 mM의 NaCl을 300 mM의 자당 3 mM의의 MgCl 2), CSKT 버퍼 (0.5 % 트리톤과 CSK 버퍼 X-100)을 각각. </li><li> cytospin이 끝나면 신속하게 빙냉 PBS로 슬라이드를 전송하고 5 분 동안 배양한다. </li><li> 얼음처럼 차가운 CSK 버퍼에 슬라이드를 전송하고 1 분 동안 품어. </li><li> 얼음처럼 차가운 CSKT 버퍼에 슬라이드를 전송하고 5 분 동안 품어. </li><li> 얼음처럼 차가운 CSK 버피로 다시 슬라이드로 이동FER과 1 분 동안 품어. </li><li> PBS로 4 % 파라 포름 알데히드로 슬라이드 이동하고, 실온에서 10 분 동안 배양한다. </li></ol> 3. 면역 형광 <ol><li> 몇 초 동안 (0.1 % 트윈 20 1X PBS) PBST와 코 플린 항아리에 슬라이드를 놓습니다. </li><li> 슬라이드 아래로 흘러 버퍼를 남아있게 경사에 슬라이드를 놓습니다. 조심스럽게 슬라이드에 묻은 액체를 닦아 물 빈 피펫 팁 상자에 넣습니다. 커버 슬립을 부드럽게 배치 차단 용액 40 μL (1X PBS, 1 % BSA, 0.1 % 트윈 -20)로 슬라이드를 오버레이하고, 실온에서 10 분 동안 배양한다. </li><li> , 커버 슬립을 제거 차단 솔루션을 제거하고 1의 농도로 희석 히스톤 H3K27me3 차 항체의 40 μl를 추가 : (500)를 차단 솔루션에. 백 슬라이드 커버 슬립을 넣고 실온에서 30 분 동안 배양한다. 주 : 블로킹 용액의 RNase 억제제 FIS이 RNA에서 RNA를 보존하는 것이 도움이 될 수도폴리 클로 날 항체를 사용하는 H 프로토콜. </li><li> 워시 부드럽게 세척하는 동안 흔들림, 5 분마다, PBST로 가득 코 플린 단지에 2 번 밀어 넣습니다. </li><li> 슬라이드 아래로 흘러 버퍼를 남아있게 경사에 슬라이드를 놓습니다. 조심스럽게 물기를 닦아 다시 팁 상자에 슬라이드를 배치합니다. 이차 항체 (1 : 500) 40 μL와 오버레이 차단 솔루션에 희석, 커버 슬립을 배치하고 어둠 속에서 15 분 동안 품어. 다음 단계는, 항상 어두운 슬라이드를 유지한다. </li><li> 단계 3.4에서와 같이 PBST 항아리를 코 플린의 과정을 2 회 반복한다. </li></ol> 올리고 뉴클레오티드 프로브 4. RNA의 물고기 <ol><li> 4.3 단계에서 RNA의 FISH 37 ° C에서 건조에서 슬라이드를 방지하기 위해 바닥에 물을 빈 피펫 팁 상자를 미리 따뜻하게. </li><li> 슬라이드 아래로 흘러 버퍼를 남아있게 경사에 슬라이드를 놓습니다. 조심스럽게 단계 3.6에서 슬라이드에 묻은 액체를 닦아 와트와 피펫 팁 상자에 슬라이드를 배치맨 아래에있는 어. 슬라이드에 물고기 세척 버퍼 (2 × SSC 10 % 포름 아미드)의 500 μl를 추가하고, 실온에서 5 분 동안 품어. </li><li> , FISH 세척 용액을 제거 슬라이드에 올리고 뉴클레오티드 프로브의 8 μl를 추가, 커버 슬립을 배치하고 바닥에 물을 미리 예열 피펫 팁 상자에 슬라이드를 전송; 1 ~ 2 시간 동안 37 ° C에서 품어. </li><li> 물고기 세척 버퍼, DAPI와 물고기 세척 버퍼 및 배 SSC 37 ° C에서 물을 욕조에 3 미리 예열 코 플린 항아리를 놓습니다. </li><li> RNA FISH 1-2 시간 배양 한 후, 미리 예열 물고기 세척 버퍼에 덮개를 씌우고. </li><li> 커버 슬립 슬라이드 오프로 떨어질 때까지 몇 분 동안 슬라이드를 품어. </li><li> 다시 세척 버퍼에 슬라이드를 놓고 5 분 동안 배양을 계속합니다. </li><li> 0.5 NG / ㎖ DAPI로 미리 예열 물고기 세척 버퍼와 다른 코 플린 항아리에 슬라이드를 전송하고 5 분 동안 품어. </li><li> 미리 예열 배 SSC와 다른 코 플린 항아리에 슬라이드를 전송하고 5 마일 부화N. </li><li> 슬라이드 아래로 흘러 버퍼를 남아있게 경사에 슬라이드를 놓습니다. 조심스럽게 물기를 닦아 건조 팁 상자에 다시 슬라이드를 배치합니다. 슬라이드에 DAPI와 antifade 4 μl를 추가하고 coverslip에 배치합니다. </li><li> 매니큐어와 커버 슬립을 봉인하고 현미경으로 관찰한다. 슬라이드는 몇 달 동안 어둠 속에서 4 ° C에 저장할 수 있습니다. </li></ol>

<ol>
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F., 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=Telomeric+RNAs+mark+sex+chromosomes+in+stem+cells.">Telomeric RNAs mark sex chromosomes in stem cells.</a> Genetics. 182, 685-698 (2009).</li><li>Raj, A., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+individual+endogenous+RNA+transcripts+in+situ+using+multiple+singly+labeled+probes.">Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes.</a> Methods Enzymol. 472, 365-386 (2010).</li><li>Kimura, H., Hayashi-Takanaka, Y., Goto, Y., Takizawa, N., Nozaki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organization+of+histone+H3+modifications+as+revealed+by+a+panel+of+specific+monoclonal+antibodies.">The organization of histone H3 modifications as revealed by a panel of specific monoclonal antibodies.</a> Cell Struct Funct. 33, 61-73 (2008).</li><li>Ogawa, Y., Sun, B. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+long+noncoding+RNAs.">Molecular mechanisms of long noncoding RNAs.</a> Mol Cell. 43, 904-914 (2011).</li></ol>

No conflicts of interest declared.

이 논문에서는 H3K27me3에 대한 슬라이드 준비, XIST RNA 물고기, 면역 형광을 완료하는 데보다 5 시간 소요 빠른 면역 FISH 프로토콜을 제시 하였다. 일반적으로 RNA FISH 밤새 배양을 필요로 XIST RNA 검출에 대한 일반적인 면역 FISH 방법과 비교하여,이 프로토콜은 크게 소요되는 시간을 줄일뿐만 아니라, 올리고 뉴클레오티드 프로브를 사용하여 면역 FISH의 감도를 향상뿐만 아닙니다. ...

포유류 X 염색체 비활성화 (XCI)은 여성에서 두 개의 X-염색체 중 하나가 전사적으로 1 불 활성화 된 것을 특징으로 XX와 XY 사이의 X-연결된 유전자 복용량 불균형을 보상하기위한 전략이다. X 염색체 불 활성화 긴 비 코딩 RNA (lncRNA) 연구를위한 훌륭한 모델 시스템입니다. X 염색체 비활성화 여러 lncRNAs 의해 규제되고, 광범위 lncRNAs, 전사, 염색질 구조와 핵 조직 (2, 3) 사이의 크로스 토크 메커니즘을 밝히기 위해 지난 수십 년 동안 연구되어왔다. X 염색체에 위치한 X 불활 센터 (XIC)의 비 RNA를 코딩하는 유전자의 제조 (4)의 개수로 이루어지는 복합 유전 궤적이다. X-비활성 특정 성적 증명서 (XIST) lncRNA eutherian 포유류는 X 염색체 불 활성화 5,6에서 중요한 역할을 하나의 lncRNA이다. XIST 성적 증명서는 쿵푸의 위치를​​ 둘러싸고진짜야 사이는 X 염색체 비활성화를 시작하고, RNA FISH를 사용하여 시각 때 구름으로 표시합니다; 이 형성 &quot;XIST 클라우드&quot;(7)라고합니다. XIST RNA는 다양한 염색질 수정 효소와 상호 작용하기 때문에, 침묵 크로 마틴과 억압 전사에 대해 서로 다른 후성 유전 학적 수정을 XIST 구름의 공동 현지화는 X 염색체 불 활성화 (8) 동안 관찰된다. 예를 들어, XIST RNA는 H3K27me3에 대한 책임과 억압 염색질 상태 (9)을 유도 polycomb 억압 복잡한 2 (PRC2)와 상호 작용합니다. 사이와 집중 H3K27me3 수정과의 공동 현지화에 XIST 구름의 발생 사이 (10, 11)의 통성 이질 염색질 풍경을 나타냅니다. 이러한 DNA / RNA FISH 등의 세포 유전 학적 기술은 향상된 감도와 최근의 진보 된 기술에 방사성 동위 원소를 프로브 (12)를 사용하여 전통적인 방법에서 먼 길을왔다여러 올리고 뉴클레오티드 프로브 (13, 14)를 사용하여 형광 이미징. 면역 형광과 함께 DNA / RNA의 물고기는 정기적으로 시공간 핵 조직, RNA 현지화, 크로 마틴 구조 및 수정을 이해하는 세포 학적 도구로 사용되어왔다. RNA FISH에 대한 대부분의 표준 프로브 준비 플라스미드 또는 박테리아 인공 염색체 (BAC) 클론 닉 번역 또는 임의 프라이밍 (15) 중 하나를 자신의 다음 라벨의 사용을 포함한다. 그러나 생쥐의 유전자의 약 70 %와 인간 유전자의 40 %는 센스 및 안티센스 성적 증명서를 구별하기 위해, 따라서 가닥 특정 FISH 방법을 필요로 센스 및 안티센스 성적 증명서 (16)의 중첩을 보여줍니다. 시험 관내 전사 RNA 프로브를 (리보 프로브 (riboprobe)를 )는 종종 특정 가닥 RNA의 FISH (17, 18)에 사용된다; 그러나, 이는 T7, SP6, 또는 T3 프로모터와 리보 프로브 합성 플라스미드 클론 또는 PCR 산물을 준비하는 것을 포함한다. 또한, genom에서 파생 된 리보 프로브IC DNA 또는 cDNA를 종종 높은 배경 노이즈가 아닌 특정 지역과 반복적 인 요소가 포함되어 있습니다. 또 다른 문제는, 길이 수백 뉴클레오타이드 복수 형광체를 포함하는 리보 프로브를 효율적으로 핵으로 침투 할 수 없다는 것이다. 이를 회피하기 위해, 하나의 단부에서 복수의 형광 물질로 표지 된 올리고 뉴클레오티드 프로브 짧아 양호한 감도, 신호 강도가 균일하고, 정제의 용이성 및 14 취급이 개발되었다. 또한, DNA 올리고 뉴클레오티드는 일반적으로 RNA보다 더 안정하다. 우리는 X 염색체 불 활성화 과정 XIST lncRNA 의해 유도 후생 유전 학적 사이의 역학 관계를 이해하기 위해 면역 형광 XIST RNA FISH와 19의 올리고 뉴클레오티드를 사용하여 유사한 전략을 적용 하였다. 이 프로토콜은 올리고 뉴클레오티드 프로브 및 세포의 적절한 제제의 창조뿐만 아니라 면역 형광 및 RNA FISH의 이용을 설명한다. 여러 oligonucle를 사용 XIST RNA FISHXIST RNA 물고기가 하나의 실험실에서 일상적으로 수행되는 경우 otides은 장기적으로 비용 효과적인 방법입니다. 이 기술은 후생 유전 학적 변형을 동시에 또는 인자와의 공동 현지화를 맵핑하는 동안 세포에서 lncRNAs를 식별하는데 사용될 수있다. 프로토콜의 하나의 큰 장점은 쉽게 하나의 연구 분야에 맞도록 수정할 수있는 기능입니다.

<table><tbody><tr><td>oligonucleotides with 5&amp;rsquo;-amino modification</td><td>IDT</td><td></td><td></td></tr><tr><td>Alexa Fluor 488 Reactive Dye</td><td>Life Technologies</td><td>A32750</td><td></td></tr><tr><td>MicroSpin G-25 columns</td><td>GE Healthcare</td><td>27-5325-01</td><td></td></tr><tr><td>Cytospin 2</td><td>Shandon</td><td>59900102</td><td></td></tr><tr><td>Bovine Serum Albumin, Molecular Biology Grade (for hybridization buffer)</td><td>Roche</td><td>10715859103</td><td></td></tr><tr><td>Histone H3K27me3 antibody</td><td>Active Motif</td><td>61017</td><td></td></tr><tr><td>Alexa Fluor 555 Goat Anti-Mouse, highly cross-adsorbed</td><td>Life Technologies</td><td>A21424</td><td></td></tr><tr><td>ProLong Gold antifade reagent</td><td>Life Technologies</td><td>P36931</td><td></td></tr></tbody></table>

quick fluorescent in situ hybridization protocol for xist rna combined with immunofluorescence of histone modification in x-chromosome inactivation

면역 형광 (면역 FISH) 동일계 하이브리드 화 (FISH)에서 RNA 형광체를 결합 단백질, 후생 유전 학적 변형 및 다른 세부 사항들의 위치 파악에 동시 통찰력 RNA 현지화 공간적 동역학을 검출하는 단일 세포 수준에서 사용될 수있는 기술을 만들어 이는 면역에 의해 강조 할 수있다. X 염색체 불 활성화 긴 비 코딩 RNA (lncRNA) - 매개 유전자 침묵에 대한 패러다임이다. 포유류 암컷의 두 X-염색체 중 하나 (XIST 구름이라고도 함) X-비활성 특정 성적 증명서 (XIST) lncRNA 축적은 X 염색체 비활성화를 시작하는 중요한 단계입니다. XIST RNA를 직접 또는 간접적으로 다양한 염색질 수정 효소와 상호 작용 및 비활성 X 염색체 (사이)에 별개의 후생 유전 학적 풍경을 소개합니다. 사이의 하나 알려진 후생 유전 학적 특징은 히스톤 H3 트리메틸 라이신 27 (H3K27me3) 수정이다. 여기서 우리는 간단하고 빠른 면역 FISH를 설명XIST RNA의 현지화와 관련된 후성 유전 적 변형을 검사 H3K27me3의 면역과 함께 여러 올리고 뉴클레오티드 프로브 RNA FISH를 사용 XIST RNA를 검출하기위한 프로토콜입니다. 올리고 뉴클레오티드 프로브 짧은 배양 시간에 결과와 시험 관내 전사 RNA 프로브 (리보 프로브)에 비해 XIST RNA의 민감한 검출 기능의 사용. 이 프로토콜은 lncRNAs의 역학 및 관련 후성 ​​유전 학적 변형, 크로 마틴 구조, 핵 조직 및 전사 조절을 이해하기위한 강력한 도구를 제공합니다.

빠른 면역 FISH의 대표 이미지는 그림 1A에 표시됩니다. 사이에 XIST RNA 구름과 H3K27me3 신호의 공동 현지화 여성 세포를 분화 검출되었다. 분화시, 12 일, EB 세포의 90 % 이상 XIST 클라우드 (도 1b)를 가지고 있었다. (150을 97 %, N = 그림 1C) 짧은 올리고 뉴클레오티드 프로브를 효율적으로 거의 모든 H3K27me3 신호 XIST RNA와 공동으로 현지화의 시각화로 이어지는 핵에 침투...

Watch this Scientific Journal Video about Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation at JoVE.co

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.

빠른 형광<em&gt; 현장에서</emX 염색체 불 활성화에 히스톤 수정의 면역 형광와 XIST RNA 결합에 대한&gt; 하이브리드 프로토콜

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell ...

quick-fluorescent-situ-hybridization-protocol-for-xist-rna-combined

Universidad de Cartagena UDEC

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JoVE Journal

Biology

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

13.9K 조회수.  Cincinnati Children's Hospital Medical Center. We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level. 

비디오: Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

이해 간체를 사용하여 초기 형성기<em&gt; 현장에서</em에서&gt; 하이브리드 프로토콜<em&gt; 아프리카 발톱 개구리</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

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Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

Prometaphase 팔 응집력과 분열의 상태를 모니터링 하려면 형광 제자리에서 교 잡 (물고기)를 사용 하 여 나 초파리 Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Zebrafish 전체 마운트 고해상도 더블 형광등 현장에서 하이브 리다이 제이션

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

생물학

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including genetic disorders, of which chromosomal abnormalities are the most common. Fluorescence in situ hybridization (FISH) is a well-known and frequently used method to detect chromosomal aberrations in humans. FISH is mainly used for the analysis of chromosomal abnormalities in the spermatozoa of males with numerical or structural chromosomal aberrations. Furthermore, this technique is also frequently applied in females to detect X chromosomal aberrations that are known to cause ovarian dysgenesis. However, information on the X chromosomal content of ovarian cells from females with X chromosomal aberrations in lymphocytes and/or buccal cells is still lacking.
The aim of this study is to advance basic research regarding X chromosomal aberrations in females, by presenting two methods based on FISH to identify the X chromosomal content of ovarian cells. First, a method is described to determine the X chromosomal content of isolated ovarian cells (oocytes, granulosa cells, and stromal cells) in non-grafted ovarian cortex tissue from females with X chromosomal aberrations. The second method is directed at evaluating the effect of chromosomal aberrations on folliculogenesis by determining the X chromosomal content of ovarian cells of newly formed secondary and antral follicles in ovarian tissue, from females with X chromosomal aberrations after long-term grafting into immunocompromised mice. Both methods could be helpful in future research to gain insight into the reproductive potential of females with X chromosomal aberrations.

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

This article presents two methods based on fluorescence in situ hybridization to determine the X chromosomal content of ovarian cells in non-grafted and grafted ovarian cortex tissue from females with X chromosomal aberrations.

Fluorescence In Situ Hybridization을 사용한 난소 세포의 X 염색체 이상 탐색

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including ...

유전학

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

제노푸스 라에비스의 신경 전구체 특성화를 위한 형광 칼슘 이미징 및 후속 시토 혼성화

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

더블 형광 결합<em&gt; 현장에서</em&gt; 마우스 뇌 섹션에서 세 가지 유전자의 발현의 검출을위한 Immunolabelling와 하이브리드

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

신경과학

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

KSHV 감염 세포에서 특정 유전자 제품의 시투 혼성화 (FISH) 및 면역 형광 (IF)의 정량형 형광

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

면역학 및 감염병학

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

전체 마운트 RNA 형광<em&gt; 현장에서</em의&gt; 하이브리드<em&gt; Drosophila</em&gt; 태아

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

결합 된 DNA-RNA 형광<em&gt; 현장에서</em&gt; 하이브리드 화 (물고기) 차별화 된 여성 마우스 배아 줄기 세포에서 X 염색체의 불 활성화를 연구하는

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

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이해 간체를 사용하여 초기 형성기

Prometaphase 팔 응집력과 분열의 상태를 모니터링 하려면 형광 제자리에서 교 잡 (물고기)를 사용 하 여 나 초파리 Oocytes

Zebrafish 전체 마운트 고해상도 더블 형광등 현장에서 하이브 리다이 제이션

Fluorescence In Situ Hybridization을 사용한 난소 세포의 X 염색체 이상 탐색

제노푸스 라에비스의 신경 전구체 특성화를 위한 형광 칼슘 이미징 및 후속 시토 혼성화

더블 형광 결합

KSHV 감염 세포에서 특정 유전자 제품의 시투 혼성화 (FISH) 및 면역 형광 (IF)의 정량형 형광

전체 마운트 RNA 형광

결합 된 DNA-RNA 형광

형광 DNA를위한 간단한 방법