Transkriptom profilering<em> In-vivo</em> Produceret Bovine præimplantationsembryoner Brug To farve Microarray Platform
Summary
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.
Abstract
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.
Introduction
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'-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.
Protocol
Representative Results
Discussion
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'et med minimum elueringsbuffer at forø…
Declarações
The authors have nothing to disclose.
Acknowledgements
Research supported by Alberta Livestock and Meat Agency, Alberta Innovates – BioSolutions, Alberta Milk, and Livestock Research Branch, Alberta Agriculture and Forestry.
Materials
| PicoPure RNA Isolation Kit | Applied Biosystems | KIT0204 | |
| RNase-Free DNase Set (50) | Qiagen | 79254 | |
| Agilent RNA 6000 Pico Kit | Agilent Technologies | 5067-1513 | |
| Arcturus RiboAmp HS PLUS Kit | Applied Biosystems | KIT0505 | |
| 2100 Bioanalyzer Instruments | Agilent Technologies | G2940CA | |
| RNA Screen Tape | Agilent Technologies | 5067-5576 | |
| ULS Fluorescent Labeling Kit | Kreatech Diagnostics | EA-021 | |
| Custom Gene Expression Microarrays | Agilent Technologies | G2514F | |
| Agilent Gene Expression wash buffer 1 | Agilent Technologies | Part #5188-5325 | |
| Agilent Gene Expression wash buffer 2 | Agilent Technologies | Part #5188-5326 | |
| 2X Hi-RPM Hybridization buffer | Agilent Technologies | Part #5190-0403 | |
| 25X Fragment buffer | Agilent Technologies | Part #5185-5974 | |
| 10X GE Blocking Agent | Agilent Technologies | Part #5188-5281 | |
| Stabilization and drying solution | Agilent Technologies | Part #5185-5979 | |
| Gasket slides enabled by Agilent SureHyb techonolgy | Agilent Technologies | G2524-60012 | Pack of 20 gasket slides, 4 microarrays/slide |
| Two-Color RNA Spike-In Kit | Agilent Technologies | Cat# 5188-5279 | |
| GenePix 4000B array scanner | Molecular Devices | GENEPIX 4000B-U | |
| Ozone Free Box | BioTray | OFB_100x200 | |
| GAL file | Agilent Technologies | – |
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