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Rapid analysis of gene expression (RAGE) facilitates universal expression profiling.

ABSTRACT: Current techniques for analysis of gene expression either monitor one gene at a time, for example northern hybridization or RT-PCR methods, or are designed for the simultaneous analysis of thousands of genes, for example microarray hybridization or serial analysis of gene expression. To provide a flexible, intermediate scale alternative, a PCR-based method for the rapid analysis of gene expression has been developed which allows expression changes to be determined in either a directed search of known genes, or an undirected survey of unknown genes. A single set of reagents and reaction conditions allows analyses of most genes in any eukaryote. The method is useful for assaying on the order of tens to hundreds of genes in multiple samples. Control experiments indicate reliable detection of changes in gene expression 2-fold and greater, and sensitivity of detection better than 1 in 10 000. Analyses of over 400 genes in a mouse system transgenic for the E2F1 gene have identified several new downstream targets of E2F1, including Brca1 and Cdk7, in addition to several unidentified genes that are upregulated in the transgenic mice. Changes in expression of several genes related to apoptosis suggest a possible potentiation of apoptotic pathways in the transgenic keratinocytes.

SUBMITTER: Wang A

PROVIDER: S-EPMC148749 | biostudies-other | 1999 Dec

REPOSITORIES: biostudies-other

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Rapid analysis of gene expression (RAGE) facilitates universal expression profiling.

Wang A A Pierce A A Judson-Kremer K K Gaddis S S Aldaz C M CM Johnson D G DG MacLeod M C MC

Nucleic acids research 19991201 23

Current techniques for analysis of gene expression either monitor one gene at a time, for example northern hybridization or RT-PCR methods, or are designed for the simultaneous analysis of thousands of genes, for example microarray hybridization or serial analysis of gene expression. To provide a flexible, intermediate scale alternative, a PCR-based method for the rapid analysis of gene expression has been developed which allows expression changes to be determined in either a directed search of ...[more]

PMID: 10556317

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Project description:BACKGROUND: Invasive aedine mosquito species have become a major issue in many parts of the world as most of them are recognised vectors or potentially involved in transmission of pathogens. Surveillance of these mosquitoes (e.g. Ae. aegypti, Yellow fever mosquito, Aedes albopictus, Asian tiger mosquito) is mainly done by collecting eggs using ovitraps and by identification of the larvae hatched in the laboratory. In order to replace this challenging and laborious procedure, we have evaluated matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) for easy and rapid species identification. METHODS: Individual protein profiles were generated using five eggs each of nine aedine species (Ae. aegypti, Ae. albopictus, Ae. atropalpus, Ae. cretinus, Ae. geniculatus, Ae. japonicus, Ae. koreicus, Ae. phoeniciae, Ae. triseriatus) from various geographical origins, and species-specific biomarker mass sets could be generated. A blinded validation using our reference data base for automated egg identification was performed. In addition, pools of 10 aedine eggs (132 two-species and 18 three-species pools) in different ratios were evaluated. RESULTS: Specific biomarker mass sets comprising 18 marker masses could be generated for eggs of nine container-inhabiting aedine species, including all the major invasive and indigenous species of Europe and North America. Two additional masses shared by all investigated aedine species are used as internal calibrators. Identification of single eggs was highly accurate (100% specificity, 98.75% sensitivity), and this method is also of value for the identification of species in pools of ten eggs. When mixing two or three species, all were identified in all pools in at least 2 or 1 of the 4 loaded replicates, respectively, if the "lesser abundant" species in the pool accounted for three or more eggs. CONCLUSIONS: MALDI-TOF MS, which is widely applied for routine identification of microorganisms in clinical microbiology laboratories, is also suited for robust, low-cost and high throughput identification of mosquito vectors in surveillance programmes. This tool can further be developed to include a wide spectrum of arthropods but also other Metazoa for which surveillance is required, and might become the method of choice for their centralised identification via online platforms.

Dataset Information

Rapid analysis of gene expression (RAGE) facilitates universal expression profiling.

Publications

Rapid analysis of gene expression (RAGE) facilitates universal expression profiling.

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