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Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells
Published on: August 5, 2016
Construction and evaluation of an ORFeome-based Brucella whole-genome DNA microarray
C Viadas1, M C Rodríguez, J M García-Lobo
1Departamento de Microbiología y Parasitología, Universidad de Navarra, Irunlarrea N1, 31008 Pamplona, Navarra, Spain. cviadas@gmail.com
Microbial Pathogenesis
|June 16, 2009
Summary
This study developed a Brucella DNA microarray using the ORFeome to analyze gene expression in Brucella abortus. The findings reveal distinct sets of highly and poorly transcribed genes, aiding in understanding Brucella pathogenesis.
Area of Science:
- Microbiology
- Genomics
- Molecular Biology
Background:
- Brucella species cause significant zoonotic diseases with major health and economic consequences.
- The availability of complete genome sequences enables high-throughput biological studies of Brucella.
- Previous work established the ORFeome of Brucella melitensis, providing a foundation for further research.
Purpose of the Study:
- To construct and validate a DNA microarray for analyzing global gene expression in Brucella.
- To determine the global transcriptional profile of Brucella abortus under laboratory conditions.
- To demonstrate the utility of ORFeome-based microarrays for microbial research.
Main Methods:
- Amplification of each Open Reading Frame (ORF) from the Brucella ORFeome.
- Printing of amplified ORFs onto coated glass slides to create a DNA microarray.
- Quality control of the microarray using image analysis and ethidium bromide staining.
- Determination of global gene expression profiles of Brucella abortus using the microarray.
- Validation of results using Reverse Transcription Polymerase Chain Reaction (RT-PCR).
Main Results:
- A Brucella DNA microarray was successfully constructed and validated.
- Two distinct sets of genes were identified: strongly and poorly expressed.
- Highly expressed genes included ribosomal proteins, Krebs cycle enzymes, and oxidative phosphorylation enzymes.
- Poorly expressed genes included virB, flagellar components, and genes associated with virulence and intracellular growth.
- Results were validated by RT-PCR, confirming the significance of the identified gene sets.
Conclusions:
- The study demonstrates the effectiveness of ORFeome-based PCR product microarrays for analyzing global gene expression in Brucella.
- This microarray approach is applicable to other microorganisms for studying gene expression.
- The findings provide a comprehensive transcriptional profile of Brucella abortus in laboratory conditions.
- The validated Brucella microarray serves as a valuable tool for studying Brucella biology and pathogenesis under various experimental conditions.
