Improving immunosensor performances using an acoustic mixer on droplet microarray

A major drawback of protein microarrays is the lack of control of ligand immobilization at the surface of the chip which limits their performances and thus their impacts in in vitro diagnosis. To improve antibody (Ab) grafting during the spotting process on commercialized gold SPRi chips, we propose to produce a chaotic flow in every spotted droplet, by using an acoustic field, in order to disrupt the steady state of the reaction of Ab grafting. Our results show that acoustic mixing during Ab binding at the biochips surface increases their biorecognition performances of a mean factor of 2.7 in comparison with Ab layer grafted in a passive mode. Moreover, it increases statistically the homogeneity of the response over all the surface of the chips.     

Direct selection of human genomic loci by microarray hybridization

We applied high-density microarrays to the enrichment of specific sequences from the human genome for high-throughput sequencing. After capture of 6,726 approximately 500-base 'exon' segments, and of 'locus-specific' regions ranging in size from 200 kb to 5 Mb, followed by sequencing on a 454 Life Sciences FLX sequencer, most sequence reads represented selection targets. These direct selection methods supersede multiplex PCR for the large-scale analysis of genomic regions.     

Analysis of crucial factors resulting in microarray hybridization failure

The factors that affect the formation and stability of DNA/DNA duplexes are complicated and still mostly unknown. In this study attempts were made to look for the crucial factor affecting hybridization failure in DNA microarray assays. A comprehensive range of factors were investigated simultaneously using a 25-mer oligonucleotide Potyvirus microarray. These included steric hindrance, direct/indirect labelling types, distance of a probe to the fluorescent labelling end, target (the DNA fragment used to hybridize with microarray probes) strand types either single strand or double strand, probes without mismatch and with different numbers of mismatch nucleotides (up to 36%) and different mismatch locations (5' end, centre and 3' end), probe GC content and T(m), secondary structures of probes and targets, different target lengths (0.277 kb to ~1.3 kb) and concentrations (0.1-30 nM). The results showed that whilst most of these known factors were unlikely to be the main causes of failed hybridization, there was strong evidence suggesting that the viral amplicon target structure is the most crucial factor. However, computing predicted target secondary structures by Mfold showed no correlation with the hybridization results. One explanation is that the predicted target secondary structures are different from the real structures. Here we postulate that the real target structure might be a combination of secondary structures resulting in a three-dimensional structure from exposure to three types of sub-structures: (1) a completely exposed linear structure to allow probes access for the successful hybridization and showing strong fluorescent signals; (2) a partially exposed structure to allow unstable binding and showing weak fluorescent signals; (3) a closed structure resulting in failed hybridization. These results are very important for microarray based studies as they not only provide an explanation for some current controversial results, but also provide potential resolution for the future studies. Due to the lack of available software for predicting the true target structure, development of microarrays should conduct an initial oligonucleotide probe selection procedure and those probes with capacity to hybridize with the target should be considered for the microarray development.     

Cooperativity of paired oligonucleotide probes for microarray hybridization assays

Synthetic DNA probes attached to microarrays usually range in length from 25 to 70 nucleotides. There is a compromise between short probes with lower sensitivity, which can be accurately synthesized in higher yields, and long probes with greater sensitivity but lower synthesis yields. Described here are microarrays printed with spots containing a mixture of two short probes, each designed to hybridize at noncontiguous sites in the same targeted sequence. We have shown that, for a printed microarray, mixed probe spots containing a pair of 30mers show significantly greater hybridization than spots containing a single 30mer and can approach the amount of hybridization to spots containing a 60mer or a 70mer. These spots with mixed oligonucleotide probes display cooperative hybridization signals greater than those that can be achieved by either probe alone. Both the higher synthesis yields of short probes and the greater sensitivity of long oligonucleotides can be utilized. This strategy provides new design options for microarray hybridization assays to detect RNA abundance, RNA splice variants, or sequence polymorphisms.     

Utilizing microarray spot characteristics to improve cross-species hybridization results

Cross-species hybridization (CSH), i.e., the hybridization of a (target) species RNA to a DNA microarray that represents another (reference) species, is often used to study species diversity. However, filtration of CSH data has to be applied to extract valid information. We present a novel approach to filtering the CSH data, which utilizes spot characteristics (SCs) of image-quantification data from scanned spotted cDNA microarrays. Five SCs that were affected by sequence similarity between probe and target sequences were identified (designated as BS-SCs). Filtration by all five BS-SC thresholds demonstrated improved clustering for two of the three examined experiments, suggesting that BS-SCs may serve for filtration of data obtained by CSH, to improve the validity of the results. This CSH data-filtration approach could become a promising tool for studying a variety of species, especially when no genomic information is available for the target species.     

Subtyping of influenza virus A hemagglutinine with hybridization microarray

An oligonucleotide microarray for influenza A hemagglutinine subtyping was presented. The number of probes for determination of each subtype hemagglutinine (H1-H13, H15, H16, pandemic flu H1N1)varied from 13 to 28. When testing of the microarray using 40 type A influenza virus isolates the hemagglutinin subtypes were unambiguously determined for 36 specimens.     

Factors influencing cDNA microarray hybridization on silylated glass slides

cDNA microarray technology is becoming the technique of choice for studying gene expression and gene expression patterns. Although experimental protocols are available, only limited methodological information on microarray manufacture, hybridization, and signal interpretation has been published. The aim of this paper is to provide more insight into the practical aspects of microarray construction and hybridization. The influence of the size, composition, and concentration of the spotted DNA fragments on the final hybridization signal and the effect of hybridization volume, sample concentration, and sample depletion have been tested and are discussed.     

Dextran sulfate provides a quantitative and quick microarray hybridization reaction

Hypoxic pulmonary hypertension (HPH) is an important pathophysiological process of a variety of cardiac and pulmonary diseases. But the mechanisms responsible for HPH are still not fully understood. The discoveries of endogenous gas signal molecules, nitric oxide (NO), and carbon monoxide (CO), have been moving the research of HPH to a new phase. Hydrogen sulfide (H2S), which is now being considered as the third new gas transmitter, was found to be possibly involved in the pathogenesis of HPH. But whether there exists an interaction between H2S and CO has not been clear in the pathogenesis of HPH. In this study, we found that H2S was significantly decreased in the pathogenesis of HPH. However, plasma CO level and the expressions of heme oxygenase (HO-1) protein and HO-1 mRNA were significantly increased. Exogenous supply of H2S could alleviate the elevation of pulmonary arterial pressure. At the same time, plasma CO level and the expressions of HO-1 protein and mRNA in pulmonary arteries were significantly increased. Whereas, exogenous supply of propargylglycine (PPG), an inhibitor of cystathionine gamma-lyase (CSE), decreased the plasma H2S content and worsened HPH. At the same time, plasma CO level and the expressions of HO-1 protein and mRNA in pulmonary arteries were decreased. The results showed that H2S could play a regulatory role in the pathogenesis of HPH through up-regulating CO/HO pathway.     

Mass transfer effects on DNA hybridization in a flow-through microarray

The goal of this study is to assess the influence of mass transfer phenomena on DNA hybridization kinetics in a flow-through, porous microarray for fast molecular testing. We present a scaled mathematical model of coupled convection, diffusion and reaction in porous media, which was used to simulate hybridization kinetics and to analyze the influence of convective transport on the reaction rate. In addition to computer simulations, we also present experimental data of hybridization collected on our microarray system for different flow rates. The results reported in this paper provide for a better understanding of the interaction between reaction and mass transfer processes during flow-through hybridization and suggest criteria for system design and optimization.     

Subtyping of influenza virus A hemagglutinin with a hybridization microarray

An oligonucleotide microarray for influenza A hemagglutinin subtyping was presented. The number of probes for the determination of each subtype of hemagglutinin (H1-H13, H15, H16, pandemic flu H1N1) varied from 13 to 28. When testing the microarray using 40 type-A influenza virus isolates, the hemagglutinin subtypes were unambiguously determined for 36 specimens.     

Comparison of different approaches for comparative genetic analysis using microarray hybridization

A robust analysis of comparative genomic microarray data is critical for meaningful genomic comparison studies. In this paper, we compare our method (implemented in a new software tool, GENCOM, freely available at ) with three commonly used analysis methods: GACK (freely available at ), an empirical cut-off value of twofold difference between the fluorescence intensities after LOWESS normalization or after AVERAGE normalization in which the fluorescence intensity is divided by the average fluorescence intensity of the entire data set. Each method was tested using data sets from real experiments with prior knowledge of conserved and divergent genes. GENCOM and GACK were superior when a high proportion of genes were divergent. GENCOM was the most suitable method for the data set in which the relationship between the fluorescence intensities was not linear. GENCOM has proved robust in an analysis of all the data sets tested.     

Development of a microarray assay that measures hybridization stoichiometry in moles

Microarray data is most useful when it can be compared with other genetic detection technologies. In this report, we designed a microarray assay format that transforms raw data into a defined scientific unit (i.e., moles) by measuring the amount of array feature present and the cDNA sequence hybridized. This study profiles a mouse reference universal RNA sample on a microarray consisting of PCR products. In measuring array features, a labeled DNA sequence was designed that hybridizes to a conserved sequence that is present in every array feature. To measure the amount of cDNA sample hybridized, the RNA sample was processed to ensure consistent dye to DNA ratio for every labeled target cDNA molecule, using labeled branched dendrimers rather than by incorporation. A dye printing assay was then performed in order to correlate molecules of cyanine dye to signal intensity. We demonstrate that by using this microarray assay design, raw data can be transformed into defined scientific units, which will facilitate interpretation of other experiments, such as data deposited at the Gene Expression Omnibus and ArrayExpress.     

Validation of S. Pombe sequence assembly by microarray hybridization.

We describe a method to make physical maps of genomes using correlative hybridization patterns of probes to random pools of BACs. We derive thereby an estimated distance between probes, and then use this estimated distance to order probes. To test the method, we used BAC libraries from Schizzosaccharomyces pombe. We compared our data to the known sequence assembly, in order to assess accuracy. We demonstrate a small number of significant discrepancies between our method and the map derived by sequence assembly. Some of these discrepancies may arise because genome order within a population is not stable; imposing a linear order on a population may not be biologically meaningful.