Miniaturized fluorescent RNA dot blot method for rapid quantitation of gene expression

BACKGROUND: RNA dot blot hybridization is a commonly used technique for gene expression assays. However, membrane based RNA dot/slot blot hybridization is time consuming, requires large amounts of RNA, and is less suited for parallel assays of more than one gene at a time. Here, we describe a glass-slide based miniaturized RNA dot blot (RNA array) procedure for rapid and parallel gene expression analysis using fluorescently labeled probes. RESULTS: RNA arrays were prepared by simple manual spotting of RNA onto amino-silane coated microarray glass slides, and used for two-color fluorescent hybridization with specific probes labeled with Cy3 and 18S ribosomal RNA house-keeping gene probe labeled with Cy5 fluorescent dyes. After hybridization, arrays were scanned on a fluorescent microarray scanner and images analyzed using microarray image analysis software. We demonstrate that this method gives comparable results to Northern blot analysis, and enables high throughput quantification of transcripts from nanogram quantities of total RNA in hundreds of samples. CONCLUSION: RNA array on glass slide and detection by fluorescently labeled probes can be used for rapid and parallel gene expression analysis. The method is particularly well suited for gene expression assays that involve quantitation of many transcripts in large numbers of samples

GeneTools – application for functional annotation and statistical hypothesis testing

BACKGROUND: Modern biology has shifted from "one gene" approaches to methods for genomic-scale analysis like microarray technology, which allow simultaneous measurement of thousands of genes. This has created a need for tools facilitating interpretation of biological data in "batch" mode. However, such tools often leave the investigator with large volumes of apparently unorganized information. To meet this interpretation challenge, gene-set, or cluster testing has become a popular analytical tool. Many gene-set testing methods and software packages are now available, most of which use a variety of statistical tests to assess the genes in a set for biological information. However, the field is still evolving, and there is a great need for "integrated" solutions. RESULTS: GeneTools is a web-service providing access to a database that brings together information from a broad range of resources. The annotation data are updated weekly, guaranteeing that users get data most recently available. Data submitted by the user are stored in the database, where it can easily be updated, shared between users and exported in various formats. GeneTools provides three different tools: i) NMC Annotation Tool, which offers annotations from several databases like UniGene, Entrez Gene, SwissProt and GeneOntology, in both single- and batch search mode. ii) GO Annotator Tool, where users can add new gene ontology (GO) annotations to genes of interest. These user defined GO annotations can be used in further analysis or exported for public distribution. iii) eGOn, a tool for visualization and statistical hypothesis testing of GO category representation. As the first GO tool, eGOn supports hypothesis testing for three different situations (master-target situation, mutually exclusive target-target situation and intersecting target-target situation). An important additional function is an evidence-code filter that allows users, to select the GO annotations for the analysis. CONCLUSION: GeneTools is the first "all in one" annotation tool, providing users with a rapid extraction of highly relevant gene annotation data for e.g. thousands of genes or clones at once. It allows a user to define and archive new GO annotations and it supports hypothesis testing related to GO category representations. GeneTools is freely available through www.genetools.n

Optimization of cDNA microarrays procedures using criteria that do not rely on external standards-5

<p><b>Copyright information:</b></p><p>Taken from "Optimization of cDNA microarrays procedures using criteria that do not rely on external standards"</p><p>http://www.biomedcentral.com/1471-2164/8/377</p><p>BMC Genomics 2007;8():377-377.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2147032.</p><p></p

Optimization of cDNA microarrays procedures using criteria that do not rely on external standards-4

<p><b>Copyright information:</b></p><p>Taken from "Optimization of cDNA microarrays procedures using criteria that do not rely on external standards"</p><p>http://www.biomedcentral.com/1471-2164/8/377</p><p>BMC Genomics 2007;8():377-377.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2147032.</p><p></p> lines NRK52E and AR42J) compared to self-self hybridization (rat cell line AR42J). The samples were hybridized to rat 15 k cDNA duplicates under six different blocking conditions including no blocker, 1000 ng poly(dA), and 25 to 1000 ng LNA dT blocker. Dye-swap and self self were performed for all blocking conditions (total of 24 hybridizations). Green-labelled samples are placed at the tail and red labelled samples at the head of the arrows

Optimization of cDNA microarrays procedures using criteria that do not rely on external standards-1

<p><b>Copyright information:</b></p><p>Taken from "Optimization of cDNA microarrays procedures using criteria that do not rely on external standards"</p><p>http://www.biomedcentral.com/1471-2164/8/377</p><p>BMC Genomics 2007;8():377-377.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2147032.</p><p></p