Neonatal screening by DNA microarray: spots and chips

Newborn screening (NBS) is a public-health genetic screening programme aimed at early detection and treatment of pre-symptomatic children affected by specific disorders. It currently involves protein-based assays and PCR to confirm abnormal results. We propose that DNA microarray technology might be an improvement over protein assays in the first stage of NBS. This approach has important advantages, such as multiplex analysis, but also has disadvantages, which include a high initial cost and the analysis/storage of large data sets. Determining the optimal technology for NBS will require that technical, public health and ethical considerations are made for the collection and extent of analysis of paediatric genomic data, for privacy and for parental consent.     

Tissue microarrays

The identification of disease-related genes is a major focus of modern biomedical research. Recent techniques, including array-based platforms for molecular profiling of disease tissues such as DNA arrays for expression profiling or matrix comparative genomic hybridization, allow for the comprehensive screening of the whole genome in a single experiment. Consequently, thousands of candidate genes have already been identified that may be linked to disease development and progression, and the process of lead discovery continues unimpeded. The evaluation of the clinical value of such leads is challenging because thousands of well-characterized tissue specimens must be analyzed. Tissue microarray (TMA) technology enables high-throughput tissue analyses to keep pace with the rapid process of lead discovery. With this technique, up to 1000 minute tissue samples are brought into an array format and analyzed simultaneously. The TMA technology is a fast, cost-effective, and statistically powerful method that will substantially facilitate translational research.     

Serodiagnosis of Lyme borreliosis with bead based immunoassays using multiplex technology

The serological diagnosis of Lyme borreliosis is accomplished by the detection of IgG and IgM antibodies specific for relevant antigens of the spirochetal pathogen Borrelia burgdorferi. Instead of the usual enzyme immune assay for screening and the Western blot technique for confirmation, bead based multiplex assays with multiple simultaneously performed distinct reactions can provide quick, automatically derived and reliable results in a single run by flow cytometer technology. The broad analytical dynamic range of assay signals and the high sensitivity and specificity of the multiplex formats allow even for a reliable use in CSF based analyses for antibody specificity index in supposed neuroborreliosis. Fluorescence intensity of the bead based reactions can be transformed into quantified values as U/ml, either for each single antigen or summed up for a group of relevant key antigens. Additionally or alternatively distinct reactions of single bead populations can be transformed to Western blot band equivalents. Internal and external quality controls with the multiplex systems show characteristic data equivalent to the conventional assay formats, so that the advantages of the multiplex assays are ready for use in the routine diagnostic laboratory.     

Microarray expression profiling of tissues from mice with uniparental duplications of Chromosomes 7 and 11 to identify imprinted genes

Microarray analysis allows the screening of thousands of identifiable genes in a single experiment. The challenge of this approach is to combine the new technology with established genetic tools to associate genes with specific biological function. In this study we have designed a screen to identify imprinted genes from mice with uniparental duplications of proximal Chromosomes (Chrs) 7 and 11, using microarray analysis. By comparing the expression patterns in embryonic and newborn tissues of maternally versus paternally inherited proximal Chrs 7 and 11, we have correctly identified four out of five known imprinted genes represented on a microarray. We have additionally identified two novel imprinted candidate genes as well as a differentially expressed clone that is a potential downstream target. Interpretation of the microarray data requires careful preparation of age- and strain-matched samples and attention to detail in tissue dissection technique. Received: 15 March 2001 / Accepted: 13 June 2001     

A Multiplex Label-Free Approach to Avian Influenza Surveillance and Serology

Influenza serology has traditionally relied on techniques such as hemagglutination inhibition, microneutralization, and ELISA. These assays are complex, challenging to implement in a format allowing detection of several types of antibody-analyte interactions at once (multiplex), and troublesome to implement in the field. As an alternative, we have developed a hemagglutinin microarray on the Arrayed Imaging Reflectometry (AIR) platform. AIR provides sensitive, rapid, and label-free multiplex detection of targets in complex analyte samples such as serum. In preliminary work, we demonstrated the application of this array to the testing of human samples from a vaccine trial. Here, we report the application of an expanded label-free hemagglutinin microarray to the analysis of avian serum samples. Samples from influenza virus challenge experiments in mallards yielded strong, selective detection of antibodies to the challenge antigen in most cases. Samples acquired in the field from mallards were also analyzed, and compared with viral hemagglutinin inhibition and microneutralization assays. We find that the AIR hemagglutinin microarray can provide a simple and robust alternative to standard methods, offering substantially greater information density from a simple workflow.     

Simultaneous detection of multiple cytokines from conditioned media and patient's sera by an antibody-based protein array system.

We have developed a novel technique for high-throughput simultaneous screening of multiple cytokine expression based on a protein array system. Our method has the advantage of showing the specificity of enzyme-linked immunosorbent assays, sensitivity of enhanced chemiluminescence (ECL), and high-throughput of microspot. In this system, the cytokine array membranes were created by spotting capture antibodies onto the membranes. The membranes were then incubated with biological samples such as conditioned media and patient's sera. The bound proteins were then recognized by biotin-conjugated antibodies and detected by horseradish peroxidase-conjugated streptavidin coupled with ECL. Experiments demonstrated that 24 cytokines from conditioned media and patient's sera could be simultaneously detected using this new approach. This methodology should allow us to develop many high-density protein array systems to detect a variety of proteins. To validate and quantitate the expression of key molecules in a wide range of samples, we have developed conditioned medium arrays to evaluate hundreds and even thousands of samples from individual cells and patients in a single microarray. The combinations of protein arrays and conditioned medium arrays or serum arrays will provide a powerful tool to identify the protein expression profiles and rapidly validate their expression in many types and numbers of samples.     

SNPstream UHT: ultra-high throughput SNP genotyping for pharmacogenomics and drug discovery

Single nucleotide polymorphism (SNP) genotyping is playing an increasing role in genome mapping, pharmacogenetic studies, and drug discovery. To date, genome-wide scans and studies involving thousands of SNPs and samples have been hampered by the lack of a system that can perform genotyping with cost-effective throughput, accuracy, and reliability. To address this need, Orrhid has developed an automated, ultra-high throughput system, SNPstream UHT, which uses multiplexed PCR in conjunction with our next generation SNP-IT tag array single base extension genotyping technology The system employs oligonucleotide microarrays manufactured in a 384-well format on a novel glass-bottomed plate. Multiplexed PCR and genotyping are performed in homogeneous reactions, and assay results are read by direct two-color fluorescence on the SNPstream UHTArray Imager. The systems flexibility enables large projects involving thousands of SNPs and thousands of samples as well as small projects that have hundreds of SNPs and hundreds of samples to be done cost effectively. We have successfully demonstrated this system in greater than 1,000,000 genotyping assays with >96% of samples giving genotypes with >99% accuracy     

Development of a comprehensive detection method for medicinal and toxic plant species

Pharmacologically active ingredients in plants can cause significant morbidity through their increasingly common use in herbal alternative medicines and dietary supplements. Monitoring consumer products for the presence of toxic plants is encumbered by the lack of rapid and specific assays. To create a sensitive, reliable, fast, and broad-spectrum assay for medicinal or toxic plant species, we tested multiplexed ligation-dependent probe amplification (MLPA), which requires partial genomic DNA sequences from species of plants that are not well represented in currently available genetic databases. Genomic DNA was obtained from 21 species of medicinal and/or toxic plants. The PCR products were amplified from these plants and cloned for sequencing. The MLPA method was successful with DNA samples from many different species. The use of a microarray to facilitate screening of potentially thousands of plants in a single assay also was successful. The combination of the specificity of the MLPA assay with the broad-scale capabilities of microarray technology should make this an especially useful tool in screening in foods and commercial herbal preparations to identify the plant compounds actually present. Other applications could potentially extend to the identification of any plant species in samples for academic botanical studies and for biodefense and forensics applications.     

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays¹. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration^2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein ⁴, protein-RNA⁵ or protein-DNA⁶ interactions.The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins⁷, DNA⁸, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.     

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.     

Peptide microarrays for detailed, high-throughput substrate identification, kinetic characterization, and inhibition studies on protein kinase A

A microarray-based mix-and-measure, nonradioactive multiplex method with real-time detection was used for substrate identification, assay development, assay optimisation, and kinetic characterization of protein kinase A (PKA). The peptide arrays included either up to 140 serine/threonine-containing peptides or a concentration series of a smaller number of peptides. In comparison with existing singleplex assays, data quality was high, variation in assay conditions and reagent consumption were reduced considerably, and assay development could be accelerated because phosphorylation kinetics were monitored simultaneously on 4, 12, or 96 arrays. PKA was shown to phosphorylate many peptides containing known PKA phosphorylation sites as well as some new substrates. The kinetic behavior of the enzyme and the mechanism of inhibition by AMP-PNP, staurosporin, and PKA inhibitor peptide on the peptide microarray correlated well with data from homogeneous assays. Using this multiplex setup, we showed that the kinetic parameters of PKA and the potency of PKA inhibitors can be affected by the sequence of the peptide substrate. The technology enables kinetic monitoring of kinase activity in a multiplex setting such as a cell or tissue lysate. Finally, this high-throughput method allows fast identification of peptide substrates for serine/threonine kinases that are still uncharacterized.     

An evaluation of commercial fluorescent bead-based luminex cytokine assays

The recent introduction of fluorescent bead-based technology, allowing the measurement of multiples analytes in a single 25-50 microl sample has revolutionized the study of cytokine responses. However, such multiplex approaches may compromise the ability of these assays to accurately measure actual cytokine levels. This study evaluates the performance of three commercially available multiplex cytokine fluorescent bead-based immunoassays (Bio-Rad's Cytokine 17-plex kit; LINCO Inc's 29-plex kit; and RnD System's Fluorokine-Multi Analyte Profiling (MAP) base kit A and B). The LINCO Inc kit was found to be the most sensitive assay for measuring concentrations of multiple recombinant cytokines in samples that had been spiked with serial dilutions of the standard provided by the manufacturer, followed respectively by the RnD Fluorokine-(MAP) and Bio-Rad 17-plex kits. A positive correlation was found in the levels of IFN-gamma measured in antigen stimulated whole blood culture supernatants by the LINCO Inc 29-plex, RnD Fluorokine-(MAP) and RnD system IFN-gamma Quantikine ELISA kits across a panel of controls and stimulated samples. Researchers should take the limitation of such multiplexed assays into account when planning experiments and the most appropriate use for these tests may currently be as screening tools for the selection of promising markers for analysis by more sensitive techniques.     

DNA microarrays--perspective of application for drug effectivity and safety evaluation

Microarray technology provides a unique tool for the determination of gene expression at the level of messenger RNA (mRNA). Microarray has been successfully applied to the high throughput simultaneous expression of many thousands of genes in a single experiment. One important application of DNA microarray technology, within the context of drugs effectiveness and safety evaluation studies, is its use as a screening tool for the identification of biochemical pathways, potential targets for novel molecular therapeutics, for the identification of molecular mechanisms of toxicity and to understand and predict individual drug sensitivity and resistance. The purpose of this review is presentation of the utility of DNA microarray technology in all phases of the drug discovery process.     

Estimation of relative allele frequencies of single-nucleotide polymorphisms in different populations by microarray hybridization of pooled DNA

Single-nucleotide polymorphisms (SNPs) are considered useful polymorphic markers for genetic studies of polygenic traits. A new practical approach to high-throughput genotyping of SNPs in a large number of individuals is needed in association study and other studies on relationships between genes and diseases. We have developed an accurate and high-throughput method for determining the allele frequencies by pooling the DNA samples and applying a DNA microarray hybridization analysis. In this method, the combination of the microarray, DNA pooling, probe pair hybridization, and fluorescent ratio analysis solves the dual problems of parallel multiple sample analysis, and parallel multiplex SNP genotyping for association study. Multiple DNA samples are immobilized on a slide and a single hybridization is performed with a pool of allele-specific oligonucleotide probes. The results of this study show that hybridization of microarray from pooled DNA samples can accurately obtain estimates of absolute allele frequencies in a sample pool. This method can also be used to identify differences in allele frequencies in distinct populations. It is amenable to automation and is suitable for immediate utilization for high-throughput genotyping of SNP.     

ELISA microarray technology as a high-throughput system for cancer biomarker validation

A large gap currently exists between the ability to discover potential biomarkers and the ability to assess the real value of these proteins for cancer screening. One major challenge in biomarker validation is the inherent variability in biomarker levels. This variability stems from the diversity across the human population and the considerable molecular heterogeneity between individual tumors, even those that originate from a single tissue. An additional challenge with cancer screening is that most cancers are rare in the general population, meaning that assay specificity must be very high. Otherwise, the number of false positives will be much greater than the number of true positives. Due to these challenges associated with biomarker validation, it is necessary to analyze thousands of samples in order to obtain a clear idea of the utility of a screening assay. Enzyme-linked immunosorbent assay (ELISA) microarray technology can simultaneously quantify levels of multiple proteins and, thus, has the potential to accelerate validation of protein biomarkers for clinical use. This review will discuss current ELISA microarray technology and potential advances that could help to achieve the reproducibility and throughput that are required to evaluate cancer biomarkers.     

Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human immunodeficiency type-1 viruses in human blood samples

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.     

Using DNA microarrays to study natural variation

The emerging field of genomics examines the relationship between genetic and phenotypic variation by describing and analyzing patterns of natural variation on a genome-wide scale. In this endeavor, an important tool is the use of microarrays, which enable simultaneous screening of thousands of assays. Microarrays were originally designed for the detection of differences between samples and are thus ideally suited to high-throughput studies of natural variation. Novel microarray platforms enable the high throughput survey of variation at multiple levels, including DNA sequences, gene expression, protein binding, and methylation. However, most microarray data analysis tools, notably normalization methods, were developed for experiments in which only few features differed between samples. In studies of natural variation, this assumption does not always hold, raising a number of new challenges.