Name: transcriptome profiling of in-vivo produced bovine pre-implantation embryos using two-color microarray platform
Uploaded: 2017-01-30T13:00:00
Description: Watch this Scientific Journal Video about Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform at JoVE.com

Research supported by Alberta Livestock and Meat Agency, Alberta Innovates - BioSolutions, Alberta Milk, and Livestock Research Branch, Alberta Agriculture and Forestry.

Dyret del af denne undersøgelse blev udført på det metaboliske Forskningsenheden for University of Alberta, Edmonton, Canada, med alle animalske eksperimentelle procedurer godkendt (Protokol # AUP00000131) ved University of Alberta Animal Care og brug Udvalg, og dyr passet efter til den canadiske Råd Animal Care retningslinjer (1993). 1. Embryo Produktion, Isolering af Total RNA og DNase Behandling <ol><li> For dyr eksperimentelle protokoller og embryonindsamlingsteam refererer i vores tidligere udgivelser...

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Det første problem at udføre microarray analyse ved hjælp Dag 7 bovine embryoner er ikke at få tilstrækkelige mængder af høj kvalitet RNA til at studere genekspression. Traditionelle phenol / chloroform RNA ekstraktion og ethanolfældning metode anbefales ikke til dag 7 embryoner, hvilket resulterer i lavt udbytte og mulige sidesten phenol hæmme RNA amplifikationsreaktion. I stedet en standard kolonne-baserede metode er bedre at isolere total RNA og derefter eluere RNA&#39;et med minimum elueringsbuffer at forø...

Tidlig embryonale tab i høje-producerende malkekøer er en af de største udfordringer i mejeriindustrien 1, 2. Bovin er blevet en interessant model til at studere human preimplantation embryo udvikling på grund af deres lignende udviklingsproces 3, 4. Men der kræves mere forskning for at få en bedre forståelse om gener involveret i kvæg tidlig fosterudvikling. Efter tyve år siden den første microarray teknologi udviklet i 1995 5, udvikling af mere sofistikerede sonde fabrikation teknologi reducerede trykfejl og variabilitet-chips inden for og mellem forskellige microarray platforme 6. Forbedret microarray teknologi resulterede i en udbredt anvendelse af denne teknologi i klinisk forskning 7 og senest i begyndelsen af embryo qVALITET vurdering 8. Den store mængde af nødvendige materiale til microarray teknologi er den vigtigste grund til, at microarray teknologi oprindeligt undladt at indtaste en række forskningsfelter såsom tidlige fosterudvikling. For nylig er RNA amplifikationsmetoder blevet forbedret til lineært amplificere RNA op til mikrogram niveau fra sub-nanogram udgangsmateriale RNA materiale 9. Der er flere kommercielle RNA forstærkning kits til rådighed på markedet; imidlertid er mere populære veludviklede kits relateret til Ribo-Single Primer Isoterm Amplification 10 og T7 promoter drevet 11 metoder. Den mest populære antisense-RNA-amplifikation anvender in vitro transkription med en oligo dT primer linker til en T7-promotor ved 5&#39;-enden 12. Denne teknologi gør det muligt at opretholde den mest repræsentative anti-sense udskrifter efter lineær forstærkning feller arrays hybridisering 13. Denne metode er blevet indrettet til at forstærke picogram niveau af totalt RNA ekstraheret fra bovin embryo 8. Universal Linkage System (ULS) er mærkningen metode, som direkte inkorporerer DNA eller forstærkede RNA med platin-linked fluorescerende farvestof enten Cyanin 547 eller Cyanin 647, ved at danne en koordinerende obligation på N7-positionen i guanin 14. Denne fremgangsmåde blev tilpasset med embryoner forskning for at generere mere stabilt amplificeret aRNA uden ændringer i forhold til aminoallyl modificeret aRNA genereret ved enzymatisk metode 15. Både enkelt farvestof og to farvestoffer mærkning metoder er blevet tilpasset ved hjælp af Universal Linkage System i microarray. En stor microarray sammenligninger undersøgelsen var, at der er en god korrelation af datakvaliteten mellem en- og to-farvet array-platforme 6. For nylig, både T7-promotor-drevetn antisense RNA amplifikation og ULS mærkning metoder er blevet udviklet til at give en mere pålidelig protokol til at generere en tilstrækkelig mængde af høj kvalitet mærket Arna materialer til microarray hybridisering 8, 16. Derfor er denne undersøgelse giver en protokol til at demonstrere nogle af de vigtige skridt fra RNA ekstraktion til dataanalyse involveret i tofarvede microarray hjælp Dag 7 bovine embryoner som eksempel.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PicoPure RNA Isolation Kit</td>
			<td>Applied Biosystems</td>
			<td>KIT0204</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-Free DNase Set (50)</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent RNA 6000 Pico Kit</td>
			<td>Agilent Technologies</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>Arcturus RiboAmp HS PLUS Kit</td>
			<td>Applied Biosystems</td>
			<td>KIT0505</td>
			<td></td>
		</tr>
		<tr>
			<td>2100 Bioanalyzer Instruments</td>
			<td>Agilent Technologies</td>
			<td>G2940CA</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Screen Tape</td>
			<td>Agilent Technologies</td>
			<td>5067-5576</td>
			<td></td>
		</tr>
		<tr>
			<td>ULS Fluorescent Labeling Kit</td>
			<td>Kreatech Diagnostics</td>
			<td>EA-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom Gene Expression Microarrays</td>
			<td>Agilent Technologies</td>
			<td>G2514F</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Agilent Gene Expression wash buffer 1</td>
			<td>Agilent Technologies</td>
			<td>Part #5188-5325</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent Gene Expression wash buffer 2</td>
			<td>Agilent Technologies</td>
			<td>Part #5188-5326</td>
			<td></td>
		</tr>
		<tr>
			<td>2X Hi-RPM Hybridization buffer</td>
			<td>Agilent Technologies</td>
			<td>&#160;Part #5190-0403</td>
			<td></td>
		</tr>
		<tr>
			<td>25X Fragment buffer</td>
			<td>Agilent Technologies</td>
			<td>Part #5185-5974</td>
			<td></td>
		</tr>
		<tr>
			<td>10X GE Blocking Agent</td>
			<td>Agilent Technologies</td>
			<td>Part #5188-5281</td>
			<td></td>
		</tr>
		<tr>
			<td>Stabilization and drying solution</td>
			<td>Agilent Technologies</td>
			<td>&#160;Part #5185-5979</td>
			<td></td>
		</tr>
		<tr>
			<td>Gasket slides enabled by Agilent SureHyb techonolgy</td>
			<td>Agilent Technologies</td>
			<td>G2524-60012</td>
			<td>Pack of 20 gasket slides, 4 microarrays/slide</td>
		</tr>
		<tr>
			<td>Two-Color RNA Spike-In Kit</td>
			<td>Agilent Technologies</td>
			<td>&#160;Cat# 5188-5279</td>
			<td></td>
		</tr>
		<tr>
			<td>GenePix 4000B array scanner</td>
			<td>Molecular Devices</td>
			<td>GENEPIX 4000B-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Ozone Free Box</td>
			<td>BioTray</td>
			<td>OFB_100x200</td>
			<td></td>
		</tr>
		<tr>
			<td>GAL file</td>
			<td>Agilent Technologies</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

transcriptome profiling of in-vivo produced bovine pre-implantation embryos using two-color microarray platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Et repræsentativt resultat af total RNA og amplificeret aRNA fra dag 7 bovine embryoner er vist i figur 3 og opsummeret i tabel 1. RNA integritet og profil kan vurderes efter RNA ekstraktion. Kvalitetsvurdering af RNA kunne ske ved Bioanalyzer instrument (figur 3A) og kun de prøver med RIN værdi højere end 7,0 er kvalificeret til at blive anvendt til amplifikation ...

Watch this Scientific Journal Video about Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform at JoVE.com

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Transkriptom profilering<em&gt; In-vivo</em&gt; Produceret Bovine præimplantationsembryoner Brug To farve Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Research

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Developmental Biology

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

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Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyserer<em&gt; In vivo</em&gt; Cell Migration hjælp Cell transplantationer og Time-lapse Imaging i zebrafisk embryoner

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

The Power of Simplicity: Søpindsvin embryoner som<em&gt; I Vivo</em&gt; Developmental modeller til undersøgelser Complex celle-til-cellesignalering netværksinteraktioner

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

In Vivo Imaging af transgene genekspression i Individuel Retinal progenitorer i kimære zebrafisk embryoner til Studieinformationen Cell Nonautonomous påvirkninger

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Induktion af hypoxi i levende frø og zebrafiskembryoner

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

Hurtig og effektiv ekspression af flere proteiner i aviær embryoner ved hjælp af mRNA-elektroporation

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Tredimensionalt Angiogeneseanalysesystem ved hjælp af co-Culture Sfæroider dannet af Endotelkoloni dannende celler og mesenchymale stamceller

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

Kvantificering af ethanolniveauer i zebrafiskembryoner ved hjælp af head space gaskromatografi

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...