Gene-specific oligonucleotide probes are currently used in microarrays to avoid cross-hybridization of highly similar sequences. We developed an approach to determine the optimal number and length of gene-specific probes for accurate transcriptional profiling studies. The study surveyed probe lengths from 25 to 1000 nt. Long probes yield better signal intensity than short probes. The signal intensity of short probes can be improved by addition of spacers or using higher probe concentration for spotting. We also found that accurate gene expression measurement can be achieved with multiple probes per gene and fewer probes are needed if longer probes rather than shorter probes are used. Based on theoretical considerations that were confirmed experimentally, our results showed that 150mer is the optimal probe length for expression measurement. Gene-specific probes can be identified using a computational approach for 150mer probes and they can be treated like long cDNA probes in terms of the hybridization reaction for high sensitivity detection. Our experimental data also show that probes which do not generate good signal intensity give erroneous expression ratio measurement results. To use microarray probes without experimental validation, gene-specific probes approximately 150mer in length are necessary. However, shorter oligonucleotide probes also work well in gene expression analysis if the probes are validated by experimental selection or if multiple probes per gene are used for expression measurement.

CRMP-1 appears to be involved in cancer invasion and metastasis and may be an invasion-suppressor gene.

No abstract

To understand the RNA expression in response to acid stress of Helicobacter pylori in genomic scale, a microarray membrane containing 1,534 open reading frames (ORFs) from strain 26695 was used. Total RNAs of H. pylori under growth conditions of pH 7.2 and 5.5 were extracted, reverse transcribed into cDNA, and labeled with biotin. Each microarray membrane was hybridized with cDNA probe from the same strain under two different pH conditions and developed by a catalyzed reporter deposition method. Gene expression of all ORFs was measured by densitometry. Among the 1,534 ORFs, 53 ORFs were highly expressed (м30% of rRNA control in densitometry ratios). There were 445 ORFs which were stably expressed (<30% of rRNA in densitometry) under both pH conditions without significant variation. A total of 80 ORFs had significantly increased expression levels at low pH, while expressions of 4 ORFs were suppressed under acidic condition. The remaining 952 ORFs were not detectable under either pH condition. These data were highly reproducible and comparable to those obtained by the RNA slot blot method. Our results suggest that microarray can be used in monitoring prokaryotic gene expression in genomic scale.Helicobacter pylori is the causative agent of chronic superficial gastritis in humans, and the presence of this organism increases risk of development of peptic ulcer disease and adenocarcinoma and mucosa-associated lymphoid tissue lymphoma of the distal stomach (2,3,10,19,21). The mechanism of pathogenesis remains largely unknown. The ability to survive under acidic stomach conditions might be one of the virulence mechanisms. Monitoring the response of H. pylori genes during acid stress may be helpful to understand the pathogenesis.Expression of many bacterial genes is induced in response to environmental stimuli (17). For technical reasons, only a limited subset of genes could be simultaneously analyzed until recently. Differential display of subsets of mRNA on a sequencing gel allows a broad search for expression differences (11, 15), but the method has been difficult to standardize. An rRNA subtraction approach has been used to identify differentially expressed genes in Mycobacterium avium (23); however, removal of rRNA by subtraction caused loss of mRNA. Chuang et al. (6) have analyzed differentially expressed genes in Escherichia coli by a method based on hybridization to spot overlapping clones. This method required subcloning and subsequent sequencing for the identification of relevant genes.Recently, a system for monitoring of a large number of gene expressions has been developed in eukaryotes using DNA microarray (5, 23, 24) or oligonucleotide microarray (16,22,29). Labeled cDNA, or in vitro-transcribed mRNA, was hybridized to the high-density probe microarray. Microarrays are able to analyze the expression of hundreds of genes in a single hybridization experiment. DNA array has also been recently adopted for monitoring gene expressions in bacteria. Unfortunately, since bacterial mRNA could not be separated from r...

DNA or oligonucleotide arrays are widely used for large-scale expression measurements, using various implementations: macroarrays in which DNA is spotted onto nylon membranes of relatively large dimensions (with radioactive detection) on the one hand; microarrays on glass slides and oligonucleotide chips, both used with fluorescent probes, on the other hand. Nylon micro-arrays with colourimetric detection have also been described recently. The small physical dimensions of miniaturized systems allow small hybridization volumes (2-100 microl) and provide high probe concentrations, in contrast to macroarrays. We show, however, that actual sensitivity (defined as the amount of sample necessary for detection of a given mRNA species) is in fact similar for all these systems and that this is mostly due to the very different amounts of target material present on the respective arrays. We then demonstrate that the combination of nylon microarrays with(33)P-labelled radioactive probes provides 100-fold better sensitivity, making it possible to perform expression profiling experiments using submicrogram amounts of unamplified total RNA from small biological samples. This has important implications in basic and clinical research and makes this alternative approach particularly suitable for groups operating in an academic context.