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 억제하실 수 있습니다. 우리는 DNA의 수량뿐만 아니라 전반적인 흡광 곡선의 모양과 280분의 260, 230분의 260 비율을 모두 측정하는 Nanodrop 분광 광도계를 사용하여 DNA의 품질을 시금 것이 좋습니다. 워싱 : 발굴 쉬운 하이브 리다이 제이션 솔루션은 매우 빠르게화될으로 하이브리드화 카세트에서 슬라이드를 제거하고 온수 세척 솔루션에 전송하면 하이브 리다이 제이션 프로세스에서 중요한 단계입니다. 그것은 씻어 솔루션 중립적인 산도에서하는 것이 중요하며 언바운드 프로브를 제거하는 동안 세척 솔루션의 온도는 엄중 중요합니다. 슬라이드를 건조 후 바로 스캔하지 않을 경우, 또는 나중에 그들을 다시 검색하고자하는 경우 스캔 후 어둠 속에서 그들을 유지하는 것이 중요합니다. 오존 : 배열 CGH를 수행할 때 오존이 세계적인 우려를하고 있습니다. 5ppm 이상의 오존 수준은 Cye 염료를 부정 영향을 보여왔다. Cye 5 오존 저하에 특히 민감합니다. 오존 노출을 최소화하는 것은 Cye 색소 저하 및 스캔 전과 도중에 신호의 손실을 방지하는 열쇠입니다. 예를 들어 야간에 검사, 환경 오존은 일반적으로 낮은 경우 하나의 가능한 옵션입니다. 세탁 및 스캔 자동 슬라이드 및 다양한 화학 슬라이드 트리 트먼트에 대한 오존 무료 엔클로저 상업적으로 사용할 수 있습니다. 오존 저항 Cye의 염료는 또한 최근에 개발되었습니다.

<table><tbody><tr><td>5X Klenow Buffer</td><td>Reagent</td><td>Promega Corp.</td><td></td><td></td></tr><tr><td>Random Octamers </td><td>Reagent</td><td>Alpha DNA</td><td></td><td></td></tr><tr><td>10X dNTP mix</td><td>Reagent</td><td>Promega Corp.</td><td></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></td><td></td></tr><tr><td>Cy-5 labeled dCTP</td><td>Reagent</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Human Genomic DNA Reference</td><td>Reagent</td><td>Novagen, EMD Millipore</td><td></td><td>Example of a possible reference</td></tr><tr><td>Klenow</td><td>Reagent</td><td>Promega Corp.</td><td></td><td></td></tr><tr><td>YM-30 Column</td><td>Reagent</td><td>EMD Millipore</td><td></td><td></td></tr><tr><td>Cot-1 DNA</td><td>Reagent</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>DIG Easy</td><td>Reagent</td><td>Roche Group</td><td></td><td></td></tr><tr><td>Sheared Herring Sperm DNA</td><td>Reagent</td><td>Promega Corp.</td><td></td><td></td></tr><tr><td>Coverslip</td><td>Reagent</td><td>Fisher Scientific</td><td></td><td>22mm x 60mm</td></tr><tr><td>Hybridization Cassette</td><td>Tool</td><td>Telechem</td><td></td><td></td></tr></tbody></table>

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 ...

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12.7K 조회수.  BC Cancer Research Centre. *The slides can remain in the 

비디오: Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to particular habitats and management practices. This article shows how certain terroir signatures can be detected in the berry metabolome and transcriptome of the grapevine cultivar Corvina using multivariate statistical analysis. The method first requires an appropriate sampling plan. In this case study, a specific clone of the Corvina cultivar was selected to minimize genetic differences, and samples were collected from seven vineyards representing three different macro-zones during three different growing seasons. An untargeted LC-MS metabolomics approach is recommended due to its high sensitivity, accompanied by efficient data processing using MZmine software and a metabolite identification strategy based on fragmentation tree analysis. Comprehensive transcriptome analysis can be achieved using microarrays containing probes covering ~99% of all predicted grapevine genes, allowing the simultaneous analysis of all differentially expressed genes in the context of different terroirs. Finally, multivariate data analysis based on projection methods can be used to overcome the strong vintage-specific effect, allowing the metabolomics and transcriptomics data to be integrated and analyzed in detail to identify informative correlations.

This article describes the application of untargeted metabolomics, transcriptomics and multivariate statistical analysis to grape berry transcripts and metabolites in order to gain insight into the terroir concept, i.e., the impact of the environment on berry quality traits.

포도 베리 대사 체학 및 Transcriptomics을 통해 해석 떼루아 개념

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to ...

연구

유전학

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including biological such as T-DNA or transposon, chemical such as ethyl methanesulfonate (EMS), or physical such as ionization radiation. The type of mutation observed varies depending on the mutagen used. For ionization radiation induced mutants, mutations include deletion, duplication, or rearrangement. While T-DNA or transposon-based mutagenesis is limited to species that are susceptible to transformation, chemical or physical mutagenesis can be applied to a broad range of species. However, the characterization of mutations derived from chemical or physical mutagenesis traditionally relies on a map-based cloning approach, which is labor intensive and time consuming. Here, we show that a high-density genome array-based comparative genomic hybridization (aCGH) platform can be applied to efficiently detect and characterize copy number variations (CNVs) in mutants derived from fast neutron bombardment (FNB) mutagenesis in Medicago truncatula, a legume species. Whole genome sequence analysis shows that there are more than 50,000 genes or gene models in M. truncatula. At present, FNB-induced mutants in M. truncatula are derived from more than 150,000 M1 lines, representing invaluable genetic resources for functional studies of genes in the genome. The aCGH platform described here is an efficient tool for characterizing FNB-induced mutants in M. truncatula.

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

This protocol provides experimental steps and information about reagents, equipment, and analysis tools for researchers who are interested in carrying out whole genome array-based comparative genomic hybridization (CGH) analysis of copy number variations in plants.

어레이 기반 비교 Genomic 교 잡 플랫폼 효율적인 탐지의 복사 번호 유사 빠른 중성자 유도 개자리속 truncatula 돌연변이에 대 한

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including ...

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid&#183;ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of &#946;-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.

In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

고밀도 DNA 및 RNA 마이크로어레이 - 광소학 합성, 하이브리드화 및 대형 핵산 라이브러리의 제조

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite ...

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

Zebrafish Oligonucleotide Microarray의 플랫폼을 사용하여 글로벌 유전자 발현 분석

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

생물학

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

인간 사전 mRNA에 매핑 Ribonucleoproteins에 대한 신속한 하이 스루풋 방법 (RNPs)

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

배열 CGH, 전체 exome 시퀀싱과 설치류에 Utero에서 Electroporation 뇌 기형에 대 한 원인 유전자를 식별 하는 새로운 전략

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of ...

신경과학

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, ...

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 ...

DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of limited use in exploring bacterial two component regulatory systems with uncharacterized activation conditions. Such systems regulate transcription only when activated in the presence of unique signals. Since these signals are often unknown, the in vitro microarray based method described in this video article can be used to determine gene targets and binding sites for response regulators. This DNA-affinity-purified-chip method may be used for any purified regulator in any organism with a sequenced genome. The protocol involves allowing the purified tagged protein to bind to sheared genomic DNA and then affinity purifying the protein-bound DNA, followed by fluorescent labeling of the DNA and hybridization to a custom tiling array. Preceding steps that may be used to optimize the assay for specific regulators are also described. The peaks generated by the array data analysis are used to predict binding site motifs, which are then experimentally validated. The motif predictions can be further used to determine gene targets of orthologous response regulators in closely related species. We demonstrate the applicability of this method by determining the gene targets and binding site motifs and thus predicting the function for a sigma54-dependent response regulator DVU3023 in the environmental bacterium Desulfovibrio vulgaris Hildenborough.

DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.

세균에 대한 두 개의 구성 요소 규제 시스템을 유전자 표적을 확인하는 DNA 친화력 - 정제 칩 (DAP-칩) 방법

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of ...

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.