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

Desideriamo ringraziare i membri della Lam Array Wan Lab squadra BAC soprattutto Miwa Suzuki e Bryan Chi per la preparazione di questo articolo. Questo lavoro è stato sostenuto da fondi dal Canadian Institutes for Health Research, Genome Canada / Genome British Columbia, e NIH / NIDCR concedere RO1 DE15965-01.

SONDA ETICHETTATURA Nota: limite di esposizione di coloranti Cye alla luce in ogni momento (questo può essere realizzato lavorando in una zona buia o schermatura dei tubi con una copertura, come un foglio di alluminio) <ol><li> Combina: (Setup 1 tubo di reazione di rinvio e 1 tubo di reazione per il campione) <ul><li> DNA (25-400 ng) </li><li> 5 ml di tampone 5X random primer (concentrazione finale: 5X Promega Klenow tampone e 7 mg / mL octamers casuale) </li><li> Diluire a 17,0 microlitri di volume totale con acqua distillata H 2 O </li></ul></li><li> Far bollire per 10 minuti a 100 ° C. Trasferire immediatamente in ghiaccio per 1 minuto. </li><li> Aggiungere 4 ml di 10X mix di dNTP (2mM dATP, dGTP, dTTP, dCTP 1,2 mm). </li><li> Aggiungi CyeDyes: <ul><li> Aggiungere 2 microlitri (2 nmoli) di Cy-3 dCTP etichettati al DNA del campione </li><li> Aggiungere 2 microlitri (2 nmoli) di Cy-5 etichettati dCTP di DNA di riferimento (ad esempio, il DNA genomico Novagen umano) </li></ul></li><li> Aggiungere 2,5 ml di Klenow (9 U / mL, Promega) e mescolare. </li><li> Incubare a 37 ° C per una notte * (~ 18 ore). * Il tempo di ibridazione durante la notte può essere regolata tra 14 a 24 ore senza danneggiare il processo di etichettatura per adattarsi a una pianificazione di laboratorio. </li></ol> Purificazione del campione (Combinato sonda pulizia e preparazione per l&#39;ibridazione) <ol><li> Utilizzando un microcontrollore YM-30 colonna: <ul><li> Aggiungere 100 ul Cot-1 DNA (1μg/μL) alla colonna. Non toccare membrana con puntale. </li><li> Piscina le reazioni (e del campione) e aggiungere alla colonna. </li><li> Colonna posto in tubo forniti e girare a 13000 g per 10 minuti. </li><li> Aggiungere 200 ul H 2 0 distillata a membrana e ripetere di selezione per lavarsi. </li><li> Gettare tubo e aggiungere 45 ul di soluzione di ibridazione: (Roche DIG Easy) </li></ul> Facoltativo: aggiungere 5μL tranciati DNA degli spermatozoi di aringhe (unità 10μg/μL, Promega) <ul><li> Inverti microcontrollore, posto in un tubo di nuova etichetta, e far girare a 3000 g per 3 minuti. </li></ul></li></ol> CALCOLO delle incorporazioni <ol><li> Rimuovere 1,5 microlitri e fluoroforo incorporazione misura con spettrofotometro NanoDrop. Utilizzo del buffer di ibridazione come uno spazio (DIG Easy) seguire le indicazioni sullo schermo. (È possibile recuperare il campione dal piedistallo, se necessario, dopo la misura.) (Nota:. Incorporazioni di sotto di 3,0 pmol / mL in uno dei due canali hanno mostrato risultati variabili) </li><li> Denaturare a 85 ° C per 10 minuti. </li><li> Sonda posto a 45 ° C per 30 minuti a 1 ora (consente Cot-1 DNA ricottura.) </li></ol> ARRAY IBRIDAZIONE <ol><li> Posto 44 microlitri soluzione sonda sul coprioggetto (22 mm x 60 mm) (Fisher Scientific). Se disponibile, posizionare il coprioggetti su un vetrino più caldo o in blocco termico pre-riscaldato a 45 ° C per mantenere la temperatura. </li><li> Allineare scorrere sul coprioggetto e la soluzione della sonda, un bordo inferiore della slitta che permette il contatto con la soluzione di ibridazione. Continuare ad abbassare fino a quando il vetrino è attaccato alla diapositiva con tensione superficiale. Inverti diapositive. </li><li> Mettere il vetrino in cassetta ibridazione (Telechem), pre-riscaldato a 45 ° C e aggiungere 10 ml di acqua nella scanalatura inferiore (per il controllo dell&#39;umidità durante l&#39;ibridazione). </li><li> Seal cassetta e incubare per 36 - 40 ore a 45 ° C. </li></ol> ARRAY LAVAGGIO Nota: Tutte le soluzioni di lavaggio sono a pH 7,0 La rimozione del vetrino dalla cassetta ibridazione è critica. DIG Facile cristallizza rapidamente a temperatura ambiente. La slitta deve essere immediatamente immersi e la polizza di copertura rimossa nella soluzione di lavaggio. <ol><li> Aggiungere circa 60 ml di 0.1XSSC, 0,1% SDS (pH 7,0, 45 ° C) Lavare la soluzione di una vaschetta Coplin prima di aprire la camera di ibridazione. </li><li> Camera aperta, aggiungere diapositiva alla soluzione di lavaggio e rimuovere coprioggetto (dovrebbe scivolare via). </li><li> Lavare il vetrino 3 volte in 0.1XSSC + 0,1% SDS a 45 ° C per 1 minuto ciascuna con agitazione. </li><li> Sciacquare il vetrino 3 volte in 0.1XSSC fresca *. Non ci dovrebbero essere bolle residui del lavaggio SDS visibile. * Le slide possono rimanere nella soluzione di lavaggio finale, fino al momento di centrifuga e di scansione (~ 15 minuti). </li><li> Centrifugare il vetrino in un mL 50 tubi Falcon a 700 g per 3 minuti. </li><li> Immediatamente scansione le diapositive (intensità del segnale diminuisce nel 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>

DNA di scarsa qualità non fornirà un buon profilo di ibridazione. E &#39;essenziale assicurare che il DNA del campione e di riferimento sono esenti da contaminanti come il fenolo, RNA, sale, ecc che possono interferire con il passaggio casuale di etichettatura principale prima di iniziare un esperimento di ibridazione. Per esempio la risospensione del DNA in Tris - EDTA (TE) al posto dell&#39;acqua non è raccomandato in quanto la concentrazione di sali in grado di inibire la reazione di marcatura. Si consiglia di saggiare la qualità del...

<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

Ibridazione genomica comparativa Array (array CGH) è un metodo per rilevare gli utili e le perdite di segmenti di DNA o di dosaggio del gene nel genoma 1. I recenti progressi nella tecnologia hanno permesso questo confronto ad alta risoluzione di interi genomi per l&#39;identificazione di alterazioni genetiche nel cancro e altre malattie genetiche 2. Il Sub-Megabase risoluzione Tiling-set array (o SMRT) array è costituito da un insieme di circa 30.000 sovrapposizione cromosoma batterico artificiale (BAC) cloni che coprono il genoma umano in ~ 100 coppie kilobase (kb) segmenti 2. Questi obiettivi BAC sono individualmente sintetizzato e individuato in duplice copia su un vetrino solo 2-4. CGH Array si basa sul principio della ibridazione competitiva. Campione di DNA e di riferimento sono etichettati in modo differenziale con Cyanine-3 e coloranti fluorescenti Cyanine-5, e co-ibridato alla matrice. Dopo un periodo di incubazione i campioni non legato sono lavati dalla diapositiva e l&#39;array è ripreso. Un pacchetto disponibile gratuitamente il software personalizzato chiamato SeeGH (www.flintbox.ca) è usato per trattare i grandi volumi di dati raccolti - un singolo esperimento genera 53.892 punti dati. SeeGH visualizza il rapporto segnale log2 intensità tra i 2 campioni a ciascun target BAC che è allineata verticalmente con posizione cromosomica 5,6. L&#39;array SMRT in grado di rilevare alterazioni più piccolo di 50 kb di dimensione 7. L&#39;array SMRT in grado di rilevare una serie di eventi riarrangiamenti del DNA compresi gli utili del DNA, le perdite, amplificazioni e delezioni omozigoti. Un vantaggio unico del vettore SMRT è che si può utilizzare il DNA isolato da campioni di paraffina fissati in formalina embedded. Se combinato con i requisiti di ingresso bassa di DNA non amplificato (25-100 ng) questo permette il profiling di campioni preziosi come quelli prodotti da microdissezione 7,8. Questo è attribuito al grande formato di ciascun target ibridazione BAC, che permette il legame di sufficiente campioni etichettati in modo da produrre i segnali per il rilevamento. Un altro vantaggio di questa piattaforma è la tolleranza di eterogeneità del tessuto, riducendo la necessità di microdissezione del tessuto noioso 8. Questo protocollo video è uno step-by-step tutorial dall&#39;etichettatura il DNA di ingresso fino alla acquisizione del segnale per l&#39;intera gamma piastrelle SMRT genoma percorso.

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

Questo video è una dimostrazione tecnico del protocollo di ibridazione per intero genoma piastrelle CGH array di percorso, che analizza l&#39;intero genoma umano utilizzando solo 25-100 ng di DNA che può essere isolato da una varietà di fonti, tra cui materiale d&#39;archivio formalina fisso.

Dimostrazione tecnica di ibridazione Whole Genome Array Comparative Genomic

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 Views.  BC Cancer Research Centre. *The slides can remain in the 

Video: 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.

Il concetto Terroir interpretato attraverso uva a bacca Metabolomica e Trascrittomica

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

Ricerca

Genetica

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.

Una basata su Array Comparative Genomic ibridazione piattaforma per efficiente rilevamento di copia numero variazioni nei mutanti veloce neutrone-indotta Medicago truncatula

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.

Microarray di DNA e RNA ad alta densità - Sintesi fotolitografica, ibridazione e preparazione di grandi librerie di acido nucleico

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.

Analisi di espressione genica globale Utilizzando un Zebrafish piattaforma Microarray oligonucleotidi

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.

Un rapido ad alta velocità Metodo per ribonucleoproteine ​​Mapping (RNP) su Human pre-mRNA

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.

Una nuova strategia che unisce Array-CGH, sequenziamento dell'intero-esoma e nell'Utero elettroporazione in roditori per identificare geni causativi per le malformazioni del cervello

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

Neuroscienze

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.

DNA microarray: Esempio di controllo della qualità, ibridazione e scansione di array

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.

Schermi competitivo genomica delle Biblioteche di lievito con codice a barre

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 purificato per affinità Chip (DAP-chip) metodo per determinare obiettivi del gene per batterica, due sistemi di regolamentazione dei componenti

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.

Dimostrazione tecnica di ibridazione Whole Genome Array Comparative Genomic

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