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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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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">oligonucleotides with 5&#8217;-amine modification</td>
			<td style="width:187px;">IDT</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Alexa Fluor 488 Reactive Dye</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">A32750</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MicroSpin G-25 columns</td>
			<td style="width:187px;">GE Healthcare</td>
			<td style="width:119px;">27-5325-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cytospin 2</td>
			<td style="width:187px;">Shandon</td>
			<td align="right" style="width:119px;">59900102</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Bovine Serum Albumin, Molecular Biology Grade (for hybridization buffer)</td>
			<td style="width:187px;">Roche</td>
			<td style="width:119px;">10715859103</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Histone H3K27me3 antibody</td>
			<td style="width:187px;">Active Motif</td>
			<td style="width:119px;">61017</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Accutase</td>
			<td style="width:187px;">Innovative Cell Technologies</td>
			<td style="width:119px;">AT104</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Alexa Fluor 555 Goat Anti-Mouse, highly cross-adsorbed</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">A21424</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">ProLong Gold antifade reagent</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">P36931</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

Research

JoVE Journal

Biology

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

13.8K 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

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

조기 칙 태아의 현장 하이브리드화에서 더블 전체 산

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

연구

생물학

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

비 - 방사성 현장에서 하이브 리다이 제이션 노르웨이 스프루스에 대한 해당 프로토콜과 식물 종의 범위

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

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 ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

형광 현장에서</em인간의 정자에서> 하이브리드화 (물고기) 프로토콜

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

식물의 성적 증명서의 정확한 현지화에 대한 원위치 하이브리드화에서

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

형광에 의한 바이러스성 RNA의 탐지<em&gt; 현장에서</em&gt; 하이브리드화 (물고기)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

RNA 형광하여 단일 세포 유전자 전사의 분석<em&gt; 현장에서</em&gt; 하이브리드 (물고기)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

형광등과 결합 염색체 복제 타이밍<em&gt; 현장에서</em&gt; 하이브리드

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

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 ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em&gt; 현장에서</em전체 마운트 태아 및 세포 조직학에서&gt; 하이브리드는 해양 Elasmobranchs을위한 적응

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...