우리는이 문서의 준비 완 램 랩 BAC 배열 팀은 특히 미와 스즈키와 브라이언 치의 회원 감사하고 싶습니다. 이 작품은 건강 연구, 게놈 캐나다 / 게놈 브리티시 컬럼비아, 그리고 NIH / NIDCR 부여 RO1 DE15965 - 01에 대한 캐나다 연구소에서 자금을 지원했다.

프로브 라벨 참고 : 항상 빛을 Cye 염료의 한계 노출 (이것은 어두운 공간에서 근무하거나 같은 알루미늄 호일로 커버와 튜브를 차폐하여 얻을 수 있습니다) <ol><li> 컴바인 : (샘플에 대한 참조 1 반응 관에 대한 설정 1 반응 튜브) <ul><li> DNA (25-400 NG) </li><li> 배 무작위 primers 버퍼 5 μL (최종 농도 : 5 배 Promega Klenow 버퍼 7 μg / μL 무작위 octamers) </li><li> 소주 H 2 O와 17.0 μL 전체 볼륨을 희석 </li></ul></li><li> 100 10 분 보일 ° C. 1 분 얼음 즉시 전송합니다. </li><li> 10X dNTP 믹스 (2mM dATP, dGTP, dTTP, 1.2mM dCTP) 4 μL를 추가합니다. </li><li> CyeDyes 추가 : <ul><li> 샘플 DNA에 싸이 - 3 분류 dCTP 2 μL (2 nmoles) 추가 </li><li> 참조 DNA (예를 들어, Novagen 인간 게놈의 DNA)에 싸이 - 5 분​​류 dCTP 2 μL (2 nmoles) 추가 </li></ul></li><li> 2.5 Klenow의 μL (9 U / μL, Promega)와 혼합을 추가합니다. </li><li> 37 품어 ° C 야간 * (~ 18 시간). * 숙박 하이브 리다이 제이션 시간이 부정적인 실험실 일정에 맞게 라벨 과정에 영향을주지 않고 14~24시간 사이에 조정할 수 있습니다. </li></ol> 샘플 정리 (하이브 리다이 제이션에 대한 조사 정리 및 준비 결합) <ol><li> Microcon YM - 30 컬럼을 사용 : <ul><li> 100 μL 콧 - 1 DNA (1μg/μL) 열에 추가합니다. 피펫 팁과 멤브레인를 만지지 마십시오. </li><li> 풀 반응 (참조 및 샘플)과 열을 추가할 수 있습니다. </li><li> 10 분 1만3천g에서 제공 관과 스핀에 넣어 칼럼. </li><li> 멤브레인 200 μL 소주 H 2 0를 추가하고 씻어 스핀을 반복합니다. </li><li> 튜브를 취소 45 μL 하이브 리다이 제이션 솔루션을 추가합니다 (로체 파다 쉬운) </li></ul> 옵션 : 추가 5μL 가지각색 헤링 정자 DNA (10μg/μL 단위, Promega) <ul><li> 반전 Microcon, 새 라벨 관에서 개최하고, 3 분 3,000그램에 스핀. </li></ul></li></ol> INCORPORATIONS 계산 <ol><li> 1.5 μL와 NanoDrop 분광를 사용하여 측정 형광단의 설립을 제거합니다. 빈 (파고 쉬운)로 하이브 리다이 제이션 버퍼를 사용하면 화면에 나타나는 지시를 따릅니다. (필요한 경우는 측정 후, 받침대에서 샘플을 복구할 수 있습니다.) (참고 :. 3.0 pmol / μL 어느 채널에 아래 Incorporations 변수 결과를 보였습니다) </li><li> 85에서 변성 ° C 10 분. </li><li> 45 플레이스 프로브 ° C 30 분 1 시간 (콧 - 1 DNA가 어닐링 수 있습니다.)에 대한 </li></ol> 배열 하이브 리다이 제이션 <ol><li> coverslip에 플레이스 44 μL 프로브 솔루션 (22mm X 60mm) (피셔 과학). 가능한 경우, 슬라이드에 coverslip을 배치 따뜻한 또는 열 블록 ° C 온도를 유지하기 위해 45 미리 예열. </li><li> coverslip 및 프로브 솔루션을 통해 슬라이드, 하이브 리다이 제이션 용액과 접촉을 허용하는 슬라이드의 하단 한쪽 가장자리를 맞춥니다. coverslip이 표면 장력으로 슬라이드에 첨부 때까지 낮은 계속. 슬라이드를 반전. </li><li> 하이브 리다이 제이션 카세트 (Telechem)에 슬라이드를 삽입, 45 미리 예열 ° C, 그리고 낮은 홈 (하이브리드화 중에 습도를 제어하는​​)에 물 10 μL를 추가합니다. </li><li> 36 시일 카세트 및 부화 - 45 40시간 ° C. </li></ol> 배열 워싱 참고 : 모든 세척 솔루션 산도 7.0에 있습니다 하이브 리다이 제이션 카세트에서 슬라이드의 제거 중요합니다. 디그 쉬운 신속하게 상온에서 구체화될. 슬라이드 바로 포장되어 및 커버 슬립은 씻어 솔루션에서 제거되어야합니다. <ol><li> 약 60 0.1XSSC의 ML, 0.1 % SDS (산도 7.0, 45 ° C) 하이브리드화 챔버를 개방하기 전에 코플린 병 솔루션을 씻으십시오. 추가 </li><li> 오픈 챔버는 솔루션을 씻고 coverslip을 (그것이 밀어한다) 제거하는 슬라이드를 추가합니다. </li><li> ° C 일분 각 동요와 슬라이드에게 0.1XSSC 3 회 + 45 0.1 % SDS 씻으십시오. </li><li> 신선한 0.1XSSC에서 슬라이드 3 번이나 씻어 *. 표시 SDS 세척부터 잔여 거품이 없어야합니다. * 슬라이드 원심 분리기 및 (~ 15 분) 검사 준비가 될 때까지 최종 세척 용액에 남아있을 수 있습니다. </li><li> 3 분 700g에 50 ML 팔콘 튜브에서 슬라이드를 원심 분리기. </li><li> 즉시 (신호 강도가 시간이 지남에 따라 감소됩니다.) 슬라이드를 스캔 </li></ol>

<ol>
	<li>Lockwood, W. W., Chari, R., Chi, B., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+array+comparative+genomic+hybridization+technologies+and+their+applications+in+human+genetics.">Recent advances in array comparative genomic hybridization technologies and their applications in human genetics.</a> European Journal of Human Genetics. 14, 139-1x`48 (2006).</li><li>Ishkanian, A. S., Malloff, C. A., Watson, S. K., deLeeuw, R. J., Chi, B., Coe, B. P., Snijders, A., Albertson, D. G., Pinkel, D., Marra, M. A., Ling, V., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tiling+resolution+DNA+microarray+with+complete+coverage+of+the+human+genome.">A tiling resolution DNA microarray with complete coverage of the human genome.</a> Nature Genetics. 36, 299-303 (2004).</li><li>Watson, S. K., DeLeeuw, R. J., Ishkanian, A. S., Malloff, C. A., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+high+throughput+validation+of+amplified+fragment+pools+of+BAC+DNA+for+constructing+high+resolution+CGH+arrays.">Methods for high throughput validation of amplified fragment pools of BAC DNA for constructing high resolution CGH arrays.</a> BMC Genomics. 5, 6 (2004).</li><li>Watson, S. K., DeLeeuw, R. J., Horsman, D. E., Squire, J. A., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytogenetically+balanced+translocations+are+associated+with+focal+copy+number+alterations.">Cytogenetically balanced translocations are associated with focal copy number alterations.</a> Human Genetics. 120, 795-805 (2007).</li><li>Chi, B., DeLeeuw, R. J., Coe, B. P., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeeGH+--+a+software+tool+for+visualization+of+whole+genome+array+comparative+genomic+hybridization+data.">SeeGH -- a software tool for visualization of whole genome array comparative genomic hybridization data.</a> BMC Bioinformatics. 5, 13 (2004).</li><li>Chi, B., DeLeeuw, R. J., Coe, B. P., Ng, R. T., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MD-SeeGH:+a+platform+for+integrative+analysis+of+multi-dimensional+genomic+data.">MD-SeeGH: a platform for integrative analysis of multi-dimensional genomic data.</a> BMC Bioinformatics. 9, 243 (2008).</li><li>Coe, B. P., Ylstra, B., Carvalho, B., Meijer, G. A., Macaulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+the+resolution+of+array+CGH.">Resolving the resolution of array CGH.</a> Genomics. 89, 647-653 (2007).</li><li>Garnis, C., Coe, B. P., Lam, S. L., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+array+CGH+increases+heterogeneity+tolerance+in+the+analysis+of+clinical+samples.">High-resolution array CGH increases heterogeneity tolerance in the analysis of clinical samples.</a> Genomics. 85, 790-793 (2005).</li></ol>

불쌍한 품질 DNA 좋은 하이브리드화 프로필을 제공하지 않습니다. 그것은 샘플을 보장하기 위해 필수적이며, 참조 DNA는 페놀, RNA, 소금, 하이브 리다이 제이션 실험을 시작하기 전에 임의의 주요 상표 단계에 방해가 될 수 등으로 오염 물질에서 무료입니다. 예를 들어 트리스에서 DNA의 resuspension - 대신 물 EDTA (에틸렌 다이아 민 테트라 초산) (TE)가 높은 소금 농도로 사용하지 않는 것이 좋습니다는 상표 반응을 억?...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>5X Klenow Buffer</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Random Octamers </td>
<td>Reagent</td>
<td>Alpha DNA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10X dNTP mix</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>2mM dATP, dGTP, dTTP, 1.2mM dCTP</td>
</tr>
<tr>
<td>Cy-3 labeled dCTP</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cy-5 labeled dCTP</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Human Genomic DNA Reference</td>
<td>Reagent</td>
<td>Novagen</td>
<td>&nbsp;</td>
<td>Example of a possible reference</td>
</tr>
<tr>
<td>Klenow</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>YM-30 Column</td>
<td>Reagent</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cot-1 DNA</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DIG Easy</td>
<td>Reagent</td>
<td>Roche</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sheared Herring Sperm DNA</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Coverslip</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>22mm x 60mm</td>
</tr>
<tr>
<td>Hybridization Cassette</td>
<td>Tool</td>
<td>Telechem</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

technical demonstration of whole genome array comparative genomic hybridization

배열 비교 게놈 하이브 리다이 제이션의 (배열 CGH)은 DNA의 세그먼트 또는 게놈 1 유전자 복용량의 이득과 손실을 검출하는 방법입니다. 이 기술의 최근 발전은 암 및 기타 유전 질환 2 유전자 변경의 식별을 위해 전체 genomes의 고해상도 비교를 활성화했습니다. 서브 Megabase 해상도 기와 집합 배열 (또는 SMRT) 배열은 약 삼만은 세균 인공 염색체 (BAC) ~ 100 kilobase 쌍에서 인간 게놈 (KB) 세그먼트 2 스팬 클론을 중복 집합으로 구성되어 있습니다. 이러한 BAC 대상 개별적으로 합성하여 하나의 유리 슬라이드에 중복 2-4에서 발견됩니다. 배열 CGH 경쟁 하이브 리다이 제이션의 원칙에 따라 달라집니다. 샘플 및 참조 DNA가 differentially Cyanine - 3으로 표시하고 Cyanine - 5 형광 염료 및 배열에 공동 hybridized입니다. 잠복기 후 언바운드 샘플은 슬라이드에서 씻은되며 배열이 몇 군데 있습니다. SeeGH (www.flintbox.ca)라는 무료로 사용 가능한 맞춤 소프트웨어 패키지는 수집된 데이터의 양이를 처리하는 데 사용됩니다 - 하나의 실험 53,892 데이터 포인트를 생성합니다. SeeGH 수직 염색체 위치를 5,6에 부합되는 각 BAC 대상에서 2 샘플 사이의 log2 신호 강도 비율을 시각화. SMRT 배열 크기 7 50킬로바이트만큼 작은 변경을 감지할 수 있습니다. SMRT의 배열은 DNA의 수익, 손실, amplifications 및 homozygous 삭제를 포함한 DNA를 다시 정리하기의 다양한 행사를 검색할 수 있습니다. SMRT 배열의 독특한 장점은 하나 포르말린 고정 파라핀 임베디드 샘플에서 DNA 격리를 사용할 수있다는 것입니다. unamplified DNA (25 - 100ng)의 낮은 입력 요구 사항과 결합하면이 같은 microdissection 7,8에 의해 만들어진 것과 같은 귀중한 샘플 프로파일 수 있습니다. 이것은 충분한 레이블 샘플의 바인딩이 검출을위한 신호를 생성하실 수 있습니다 각 BAC의 하이브리드화 대상의 대형에 따라 결정됩니다. 이 플랫폼의 또 다른 장점은 지루한 조직 microdissection 8의 필요성을 감소, 조직 이질의 오차이다. 이 비디오 프로토콜은 전체 게놈의 기와 경로 SMRT 배열에 대한 인수를 신호까지 입력 DNA를 라벨의 단계별 자습서입니다.

Watch this Scientific Journal Video about Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization at JoVE.com

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

이 비디오 아카이브 포르말린 고정 재료를 포함한 다양한 소스에서 격리 수있는 DNA의 단지 25-100 NG를 사용하여 전체 인간 게놈을 검사 전체 게놈 기와 경로 배열 CGH에 대한 하이브 리다이 제이션 프로토콜의 기술 데모입니다.

전체 게놈 배열 비교 게놈 하이브 리다이 제이션의 기술의 데모

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

technical-demonstration-whole-genome-array-comparative-genomic

Research

JoVE Journal

Biology

12.6K 조회수.  BC Cancer Research Centre. *The slides can remain in the 

비디오: Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

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

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 ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

전체 마운트를 사용하여 현장에서</em분자 생물학과 Organismal를 링크> 하이브 리다이 제이션

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

의 DNA Microarrays : 샘플 품질 제어, 배열 하이브 리다이 제이션 및 스캔

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

전체 산<em&gt; 현장에서</emE11.5 마우스 태아에 E8.5의&gt; 하이브리드

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Barcoded 효모 게놈 라이브러리의 경쟁력 화면

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

높은 처리량 현장에서</em태아 마우스 로우어 Urogenital 트랙트의 mRNA의 표현 패턴을 특성화> 하이브리드화 방법

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

파편을 사용하여 풀링된 장면에서 드문 게놈 변종의 탐지

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

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 ...

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.