Name: technical demonstration of whole genome array comparative genomic hybridization
Uploaded: 2008-08-05T18:06:00.000+00:00
Duration: 16 min 37 s
Description: Watch this Scientific Journal Video about Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization at JoVE.com

Queremos agradecer aos membros do Laboratório de Lam Wan equipe matriz BAC especialmente Miwa Suzuki e Bryan Chi para a preparação deste artigo. Este trabalho foi financiado com recursos do Canadian Institutes for Health Research, Canadá Genoma / British Columbia Genoma, e NIH / NIDCR conceder RO1 DE15965-01.

PROBE ROTULAGEM Nota: limite de exposição de Corantes Cye à luz o tempo todo (isso pode ser conseguido através do trabalho em uma área escura ou pela proteção dos tubos com uma tampa, como folha de alumínio) <ol><li> Combine: (Setup um tubo de reação para referência e um tubo de reação para a amostra) <ul><li> DNA (25-400 ng) </li><li> 5 mL de tampão 5X primers aleatórios (concentração final: 5X Promega Klenow tampão e 7 mg / mL octâmeros aleatório) </li><li> Diluída para 17,0 mL de volume total com H 2 O destilada </li></ul></li><li> Ferva por 10 minutos a 100 ° C. Transferir imediatamente para o gelo por 1 minuto. </li><li> Adicionar 4 mL de dNTP mix 10X (2mM dATP, dGTP, dTTP, dCTP 1,2 mm). </li><li> Adicionar CyeDyes: <ul><li> Adicione 2 mL (2 nmoles) de Cy-3 dCTP marcado com o DNA da amostra </li><li> Adicione 2 mL (2 nmoles) de Cy-5 dCTP marcado para Referência DNA (por exemplo, Novagen DNA genômico humano) </li></ul></li><li> Adicionar 2,5 mL de Klenow (9 U / mL, Promega) e misturar. </li><li> Incubar a 37 ° C durante a noite * (~ 18 horas). * O tempo de hibridização durante a noite pode ser ajustada entre 14 a 24 horas sem afetar negativamente o processo de rotulagem para se adequar a um cronograma de laboratório. </li></ol> AMOSTRA CLEAN UP (Sonda combinada de limpeza e preparação para a hibridação) <ol><li> Usando um microcontrolador YM-30 coluna: <ul><li> Adicionar 100 Cot-1 mL DNA (1μg/μL) à coluna. Não toque membrana com pipeta ponta. </li><li> Piscina as reações (de referência e amostra) e adicionar a coluna. </li><li> Lugar na coluna tubo fornecido e girar à 13.000 g por 10 minutos. </li><li> Adicionar 200 mL H 2 0 destilada e repetir a membrana de giro para lavagem. </li><li> Descartar tubo e adicionar 45 mL de solução de hibridização: (Roche DIG Fácil) </li></ul> Opcional: adicione DNA do esperma 5μL sheared arenque (unidade 10μg/μL, Promega) <ul><li> Inverter microcontrolador, coloque em um novo tubo rotulado, e girar a 3000 g por 3 minutos. </li></ul></li></ol> CÁLCULO de incorporações <ol><li> Remover 1,5 mL e incorporação medida fluoróforo usando Espectrofotômetro NanoDrop. Usando o seu tampão de hibridação como um espaço em branco (DIG Easy) siga as instruções na tela. (Você pode recuperar a amostra do pedestal, se necessário, após a medição.) (Nota:. Incorporações abaixo de 3,0 pmol / mL nos dois canais têm mostrado resultados variáveis) </li><li> Desnaturar a 85 ° C por 10 minutos. </li><li> Sonda lugar a 45 ° C por 30 minutos a 1 hora (permite Cot-1 DNA annealing). </li></ol> HIBRIDIZAÇÃO ARRAY <ol><li> Coloque 44 mL de solução sonda sobre a lamínula (22 mm mm x 60) (Fisher Scientific). Se disponível, coloque a lamínula sobre uma lâmina quente ou bloco de aquecimento pré-aquecido a 45 ° C para manter a temperatura. </li><li> Alinhar deslizar sobre lamínula e solução de sonda, uma borda inferior da lâmina permitindo contato com a solução de hibridação. Continue a diminuir até a lamela está ligado ao slide com tensão superficial. Inverter slide. </li><li> Coloque o slide em cassete hibridação (Telechem), pré-aquecido a 45 ° C, e adicionar 10 mL de água na parte inferior do sulco (para controle de umidade durante a hibridação). </li><li> Cassete Seal e incubar por 36-40 horas a 45 ° C. </li></ol> LAVAGEM ARRAY Nota: Todas as soluções de lavagem são em pH 7,0 Remoção da lâmina da cassete hibridação é crítica. DIG Fácil cristaliza rapidamente a temperatura ambiente. A lâmina deve ser imediatamente imersas ea lamínula removidos na solução de lavagem. <ol><li> Adicionar aproximadamente 60 mL de 0.1XSSC, 0,1% SDS (pH 7,0, 45 ° C) lavar solução para um frasco Coplin antes de abrir a câmara de hibridação. </li><li> Câmara aberta, adicionar slides solução para lavar e remover lamela (deve escorregar). </li><li> Lave o slide 3 vezes em 0.1XSSC + 0,1% SDS a 45 ° C por 1 minuto cada, com agitação. </li><li> Lavar o slide 3 vezes em 0.1XSSC fresca *. Não deve haver bolhas residual da lavagem SDS visível. * Os slides podem permanecer na solução de lavagem final até estar pronto para centrífuga e scan (~ 15 minutos). </li><li> Centrifugar o slide em uma tubos de 50 mL Falcon em 700 g por 3 minutos. </li><li> Verificar imediatamente os slides (intensidades de sinal irá diminuir ao longo do tempo.) </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>

Pobres DNA de qualidade não vai fornecer um perfil de hibridização bom. É essencial para garantir que a amostra de ADN de referência e estão livres de contaminantes tais como fenol, RNA, sal, etc que possam interferir com a etapa de rotulagem aleatória principal antes de iniciar um experimento de hibridização. Por exemplo, a ressuspensão de DNA em Tris - EDTA (TE) em vez de água não é recomendada como alta concentração de sal pode inibir a reação rotulagem. Recomendamos assaying qualidade do DNA usando o espectrofotômetro ...

<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

Matriz hibridização genômica comparativa (array CGH) é um método para a detecção de ganhos e perdas de segmentos de DNA ou dosagem do gene no genoma 1. Os recentes avanços nesta tecnologia permitiram a comparação de alta resolução de genomas inteiros para a identificação de alterações genéticas no cancro e outras doenças genéticas 2. O Sub-Megabase Resolução série Tiling set-(ou SMRT) matriz é composta por um conjunto de cerca de 30 mil sobreposição cromossomo artificial bacteriano (BAC) clones que abrangem o genoma humano em ~ par kilobase 100 (kb) segmentos 2. Essas metas são BAC individualmente sintetizados e manchado em duplicado por uma lâmina de vidro única 2-4. Matriz CGH é baseado no princípio de hibridização competitiva. Amostra de DNA e de referência são diferencialmente marcada com Cyanine-3 e corantes fluorescentes Cyanine-5, e co-hibridizados para a matriz. Após um período de incubação, as amostras não ligadas são lavadas a partir do slide ea matriz é impressa. Um pacote de software disponível gratuitamente personalizado chamado SeeGH (www.flintbox.ca) é usado para processar o grande volume de dados coletados - um único experimento gera 53.892 pontos de dados. SeeGH visualiza o log2 relação intensidade de sinal entre as duas amostras em cada alvo BAC, que está verticalmente alinhado com a posição cromossômica 5,6. A matriz SMRT pode detectar alterações tão pequenas quanto 50 kb de tamanho 7. A matriz SMRT pode detectar uma variedade de eventos de rearranjo do DNA DNA incluindo os ganhos, perdas, amplificações e deleções homozigótica. A única vantagem da matriz SMRT é que se pode usar DNA isolado de amostras de parafina fixado em formalina incorporado. Quando combinado com os requisitos de entrada baixa de DNA sem amplificação (25-100ng), que permite perfis de amostras preciosos como os produzidos por microdissecção 7,8. Isto é atribuído ao grande tamanho de cada alvo de hibridização BAC que permite a ligação de amostras suficientes rotulados para produzir sinais para detecção. Outra vantagem desta plataforma é a tolerância da heterogeneidade do tecido, diminuindo a necessidade de microdissecção tecido tedioso 8. Este protocolo de vídeo é um tutorial passo-a-passo da rotulagem o DNA de entrada até a aquisição de sinal para o genoma série azulejos toda SMRT caminho.

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

Este vídeo é uma demonstração técnica do protocolo de hibridização para todo o genoma azulejos caminho disposição CGH, que escaneia todo o genoma humano usando apenas 25-100 ng de DNA que podem ser isoladas a partir de uma variedade de fontes, incluindo o material de arquivo formol fixa.

Demonstração de técnicas Whole Genome hibridização genômica comparativa de matriz

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

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

Vídeo: 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.

O Conceito Terroir Interpretado através da baga da uva Metabolomics e transcriptómica

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

Genética

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.

Uma plataforma array de hibridização genômica comparativa para detecção eficiente de variações no número de cópias em Medicago truncatula mutantes induzidos por nêutrons rápidos

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.

Microarrays de DNA e RNA de alta densidade-síntese fotolitográfica, hibridação e preparação de grandes bibliotecas de ácidos nucleicos

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.

Análise da expressão global de Gene usando uma plataforma Microarray Zebrafish Oligonucleotide

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

Biologia

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.

Um método rápido de alta capacidade para Ribonucleoproteínas Mapping (RNPs) no pré-mRNA Humanos

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.

Uma nova estratégia combinando Array-CGH, todo-exome sequenciamento e eletroporação no Utero em roedores para identificar Genes causadores de malformações do cérebro

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

Neurociência

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.

Microarrays de DNA: Controle de Qualidade da amostra, Hibridação Array e Digitalização

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.

Competitiva Screens Genomic de Bibliotecas Yeast Barcoded

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.

DNA-afinidade-purificada Método Chip (DAP-chip) para determinar genes alvos para bacteriana Dois Sistemas Regulatórios componentes

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.

Demonstração de técnicas Whole Genome hibridização genômica comparativa de matriz

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