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DNA microarray technology has advanced rapidly since the first use of cDNA microarrays almost a decade ago. For gene expression studies on organisms, for which the genomes have been sequenced, cDNA microarrays are being gradually replaced by gene-specific oligonucleotide microarrays. Although, cDNA microarrays give higher signal intensity than oligonucleotide microarrays, they cannot be used for the measurement of gene-specific expression, whereas, oligonucleotide microarrays can. To obtain both a high signal intensity and specificity in gene expression measurements, gene-specific oligonucleotide probes as long as 150-mers, designed using sequence databases and algorithms to identify unique sequences of genes, are used as microarray probes. In order to achieve a high signal intensity, specificity, and accurate measurement of expression, in addition to the length and sequence of the probes, it is necessary to optimize other parameters such as the surface chemistry of the microarray slides, the addition of spacers and linkers to the probes, and the composition of the hybridization solution. [ABSTRACT FROM AUTHOR]

A multiplexed glyco-bead array method for determining the sugar-binding specificities of plant lectins was described by a bead-based flow cytometric assay. Glycopeptides with N- and O-glycans were immobilized on multiplexed beads, and the specificities of several kinds of sugar chains were measured in a single reaction. This strategy is easy, rapid, reproducible, and suitable for small samples, and allows the reliable elucidation of sugar-binding properties of lectins under identical conditions. [ABSTRACT FROM AUTHOR]

Protein microarray is a powerful tool for identifying disease biomarkers and therapeutical targets, and for systematically studying biological pathways with high efficiency. Although the protein microarray platform has been adopted by proteomic research and discovery, what remains problematic is how to maintain the activities and structures of printed proteins on slide surface. With the recent accomplishments in the R & D laboratory, now scientists around the world are able to preserve the biological functions of spotted proteins for high throughput analysis. Full Moon BioSystems (FMB) has developed general guidelines, which helps scientists efficiently prepare forward as well as reverse-phase protein microarray using FMB's proprietary polymer-coated slides, to obtain reliable and accurate array data with FMB's unique detection and analysis technology. [ABSTRACT FROM AUTHOR]

As more genomes are sequenced, challenge of rapidly unraveling the functions of genes was faced. To that end, cell microarrays have been recently described that permit transfection of thousands of nucleic acids in parallel and enable the analysis of phenotypic consequences of such perturbations. As many parameters can influence the efficiency of transfection and consequently protein expression or extinction, some important features in manufacturing cell microarrays for functional exploration of genomes were described. [ABSTRACT FROM AUTHOR]

A protocol for quantification of oligonucleotide hybridization on polymer microparticles by europium(III) ion fluorescence is described. The procedure involves modification of commercially available amino-functionalized microparticles in such a manner that oligonucleotide probes may be assembled in situ on these particles or, alternatively, they may be immobilized postsynthetically. The oligonucleotide-coated particles obtained are then used as the solid phase in a mixed-phase hybridization assay. The efficiency of hybridization is quantified with the aid of oligonucleotides tagged with a europium(III) chelate. Either, the fluorescently tagged probe is hybridized directly to a complementary particle-anchored oligonucleotide, or a sandwich-type assay set up, where a third oligonucleotide complementary both to the tagged and particle-bound probe mediates the attachment to the particles, is exploited. The number of europium(III) ions attached to the solid-phase is determined by the DELFIA® protocol, involving release of the europium(III) ions in solution and development of the fluorescence by addition of an enhancement solution. Alternatively, the fluorescence intensity of the photoluminescent chelate may be measured directly from a single particle. [ABSTRACT FROM AUTHOR]

Microarrays are important tools for high-throughput analysis of biomolecules. The use of microarrays for parallel screening of nucleic acid and protein profiles has become an industry standard. A few limitations of microarrays are the requirement for relatively large sample volumes and elongated incubation time, as well as the limit of detection. In addition, traditional microarrays make use of bulky instrumentation for the detection, and sample amplification and labeling are quite laborious, which increase analysis cost and delays the time for obtaining results. These problems limit microarray techniques from point-of-care and field applications. One strategy for overcoming these problems is to develop nanoarrays, particularly electronicsbased nanoarrays. With further miniaturization, higher sensitivity, and simplified sample preparation, nanoarrays could potentially be employed for biomolecular analysis in personal healthcare and monitoring of trace pathogens. In this chapter, it is intended to introduce the concept and advantage of nanotechnology and then describe current methods and protocols for novel nanoarrays in three aspects: (1) label-free nucleic acids analysis using nanoarrays, (2) nanoarrays for protein detection by conventional optical fluorescence microscopy as well as by novel label-free methods such as atomic force microscopy, and (3) nanoarray for enzymatic-based assay. These nanoarrays will have significant applications in drug discovery, medical diagnosis, genetic testing, environmental monitoring, and food safety inspection. [ABSTRACT FROM AUTHOR]

A method of high resolution, array-based comparative genomic hybridization is described for the mapping of copy-number changes associated with chromosomal amplifications, deletions, and translocations. The method involves the design of whole-genome or targeted, fine-tiling arrays for synthesis on a high-density digital microarray-synthesis platform. The arrays can span entire eukaryotic genomes or be targeted to specific chromosomal regions for high-resolution identification of copy-number changes and the corresponding breakpoint locations. The methods described include the bioinformatics required for array design, and the protocols for DNA fragmentation, dual-color labeling, microarray hybridization, and array scanning. The processes for data extraction, normalization, and segmentation analysis are also described. [ABSTRACT FROM AUTHOR]

Synthetic peptides are widely used for production and analysis of antibodies as well as in the study of protein modification enzymes. To circumvent the technical challenges of the existing techniques regarding peptide quantization and normalization, a new method of producing peptide arrays has been developed. This approach utilizes intein-mediated protein ligation that involves linkage of a carrier protein possessing a reactive carboxyl-terminal thioester to a peptide with an amino-terminal cysteine through a native peptide bond. Ligated protein substrates or enzymetreated samples are arrayed on nitrocellulose membranes with a standard dot-blot apparatus and analyzed by immunoassay. This technique has improved sensitivity and reproducibility, and is suitable for various peptide-based applications. In this report, several experimental procedures including epitope mapping and the study of protein modifications were described. [ABSTRACT FROM AUTHOR]

Systems biology has enjoyed explosive growth in both the number of people participating in this area of research and the number of publications on the topic. The field of systems biology encompasses the in silico analysis of high-throughput data as provided by DNA or protein microarrays. Along with the increasing availability of microarray data, attention is focused on methods of analyzing the expression rates. One important type of analysis is the classification task, for example, distinguishing different types of cell functions or tumors. Recently, interest has been awakened toward artificial neural networks (ANN), which have many appealing characteristics such as an exceptional degree of accuracy. Nonlinear relationships or independence from certain assumptions regarding the data distribution are also considered. The current work reviews advantages as well as disadvantages of neural networks in the context of microarray analysis. Comparisons are drawn to alternative methods. Selected solutions are discussed, and finally algorithms for the effective combination of multiple ANNs are presented. The development of approaches to use ANN-processed microarray data applicable to run cell and tissue simulations may be slated for future investigation. [ABSTRACT FROM AUTHOR]

Here, we provide methods for the creation of protein microarrays in microplates. The microplate consists of 96 wells with each well capable of holding a protein microarray at a spot density of up to 400 (20 × 20) individual elements. Arrays of capture monoclonal antibodies, corresponding to specific interleukins, were printed onto the bottom of the wells which had been surface activated for covalent attachment. A Biomek® 2000 laboratory automation workstation (Beckman Coulter, Inc., Fullerton, CA) equipped with a high-density replicating tool was used for printing low density 3 × 3 to 5 × 5 arrays. For higher density arrays, a microarrayer system (Cartesian PS7200, Genomic Solutions, Inc., Ann Arbor, MI) was employed. Multiple antigens were simultaneously analyzed without detectable cross-reactivity associated with capture antibody or secondary antibody interactions. Detection of interleukin antigens, spiked into cell culture media containing 10% fetal calf serum, was specific and sensitive. [ABSTRACT FROM AUTHOR]

Copy-number polymorphisms at specific genomic loci have been implicated in numerous human and animal disease phenotypes. Multiplex ligation-dependent probe amplification (MLPA) is a molecular genetic technique allowing targeted quantification of genomic copy-number changes (deletions and duplications), with potential for multiplexing up to 50 loci in one assay, and resolution down to the single nucleotide level. Modification of the MLPA technique to include Cy-labeled amplification primers permits parallel product detection by capillary electrophoresis and microarray hybridization. Detection and quantification of products by sequencespecific hybridization rather than size-specific capillary electrophoresis increases the potential for probe multiplexing possible in one assay and also allows for more flexible and efficient MLPA probe design. Protocols for the printing of synthetic oligonucleotide probe-sets for the detection of MLPA products, MLPA-probe amplification using array-compatible primers, and parallel product detection by quantitative capillary electrophoresis and microarray hybridization have been optimized. [ABSTRACT FROM AUTHOR]

The study of protein-protein and protein-DNA interactions is critical to understand biological processes. This article presents the methodology to create peptide microarrays in situ for the high-throughput screening of complex biomolecules. The in situ ink-jet peptide synthesis results in a conservation of costly reagent and amino acids, whereas providing a means to produce denser peptide arrays. A smaller amount of test sample is required to observe interaction when using these high-density peptide arrays. [ABSTRACT FROM AUTHOR]

This chapter describes methodology for the labeling, hybridization, and detection of amplicon target DNA to arrays of oligonucleotide probes attached to plastic substrates. A systematic approach to target discrimination based on both hybridization and wash stringency is provided. [ABSTRACT FROM AUTHOR]

The ability to realize simultaneous genotyping of multiple single-nucleotide polymorphisms or mutations is valuable in DNA samples from complex multigenic pathologies such as cancer. In this way, the complexity (number of hybridization units per chip) of the developed MICAM? DNA chip, and the orientation of the grafted pyrrole oligonucleotides, make it particularly well adapted to the analysis of single-nucleotide polymorphisms/mutations in multiple potential tumoral markers. The proposed genotyping methodology is based on solid-phase minisequencing, where oligonucleotides are designed to anneal immediately upstream of the polymorphism sites, and labeled dideoxynucleotides are used as substrates for polymerase extension. The developed assay was applied to the analysis of the TP53 codon 72 polymorphism on DNA from cell lines and human colorectal samples. [ABSTRACT FROM AUTHOR]

A new strain of coronavirus has caused an outbreak of severe acute respiratory syndrome (SARS) from 2002 to 2003 resulting in 774 deaths worldwide. By protein chip array profiling technology, a number of serum biomarkers that might be useful in monitoring the clinical course of SARS patients were identified. This book chapter describes how the protein chip array profiling was carried out for this study. Briefly, SARS patients' serum samples were first fractionated in Q Ceramic HyperD ion exchange sorbent beads by buffers at different pH. Serum protein fractions thus obtained were then bound onto a copper (II) immobilized metal affinity capture (IMAC30 Cu [II]) ProteinChip® Array or a weak cation-exchange (CM10) ProteinChip Array. After washing and addition of sinapinic acid, the chips were read in a Protein Biological System (PBS) IIc mass spectrometer. Ions were generated by laser shots and flied in a time of flight mode to the ion detector according to their mass over charge (m/z) ratio. The serum profiling spectra in SARS patients were acquired, baseline subtracted and analyzed in parallel with those from the control subjects by Ciphergen ProteinChip Software 3.0.2 with their peak intensities compared by a nonparametric two sample Mann-Whitney-U test. More than twelve peaks were differentially expressed in SARS patients with one at m/z of 11,695 (later identified to be serum amyloid A protein), which had increase in peak intensity correlating with the extent of SARS-coronavirus induced pneumonia as defined by a serial chest X-ray opacity score. The remaining biomarkers could also be useful in the study of other clinical parameters in SARS patients. [ABSTRACT FROM AUTHOR]

The concept of DNA arrays was first introduced in the early 1980s, by Sir Edwin Southern. Since then, many research institutions and biotechnology companies have investigated the potential use of arrays in fields ranging from genetic diagnostics to forensics investigations. A 64-channel automated chemical delivery system, known as the Southern Array Maker, which synthesizes oligonucleotides directly onto an aminated polypropylene substrate has been constructed. Many different arrays have been synthesized for the purpose of detecting single point mutations, which might be either indicators of, or directly responsible for, many different types of genetic diseases and cancers. These include cystic fibrosis, H-ras, K-ras, and other mutations. In addition to the synthesized arrays, we are also looking into various alternative methods of producing both highand low-density DNA arrays. This chapter is intended to demonstrate the synthesis of oligoarrays by in situ method using standard phosphoramidite chemistry. Phosphoramidate linkage to the aminated polypropylene is quite stable under oligo cleavage and deprotection conditions. Oligonucleotide density is approx 3 pmole or 1012 molecules/mm2. [ABSTRACT FROM AUTHOR]

Optimization of oligonucleotide DNA microarrays is a multiparametric problem. The goal of the optimization process is to get conditions that capture target DNA with high sensitivity and selectivity. Parameters determining the performance of the microarray are spot morphology, probe and target density, background, and selectivity. More than 10 variables can be adjusted to obtain a well-optimized protocol. However, some variables only affect spot morphology and other factors affect for instance hybridization and selectivity, which can limit the optimization work considerably. This chapter suggests an outline on how an optimization procedure is made. Moreover, a simple method for absolutely quantify the number of hybridized target to the spots is given. The latter is important because it gives the possibilities to compare results with those obtained in the literature. [ABSTRACT FROM AUTHOR]

Gene expression technology offers great potentials to generate new insights into human disease pathogenesis; however, the data quality remains a major obstacle for realizing its potentials. In the present study 60-mers oligonucleotide target immobilized on coated glass slides were utilized as a model system to investigate parameters, such as target concentration, retention, signal linearity, and fluorescence properties of fluorophores, which likely affect the quality of microarray results. An array calibration slide was used to calibrate an Axon GenePix 4000A scanner and ensure the dynamic range of the instrument. The work is a first step toward our goal of quantitative gene expression measurements. [ABSTRACT FROM AUTHOR]

Oligonucleotide microarray has become an important and powerful tool for various genomic analyses, where, unlike conventional methods, one can identify simultaneously a large number of targets in a given sample. Postsynthesis immobilization, the most widely used method, involves the noncovalent and covalent fixing of suitably modified oligonucleotides on the solid supports. Among the various functional groups aminoalkyl, hydroxyalkyl, mercaptoalkyl, aldehyde, epoxy, and carboxyl the most frequently used functional groups on the polymeric surfaces. Because glass and polypropylene, the most widely used materials, are nonporous in nature, the functional groups density on the surface remains very low. In order to know the exact concentration of a ligand to be immobilized, it is essential to estimate the accessible functional groups on these surfaces. For this purpose, sensitive methods are required to estimate exact density of available functional groups on the surfaces. Apart from physical methods, a number of sensitive chemical methods, by making use of high extinction coefficient of 4,4′-dimethoxytrityl cation (ε498 = 70,000 Lmol−11cm−1), have been reported to estimate accessible functional groups on the glass based polymer supports. In this chapter, use of these reagents for spectrophotometric determination of functional group density on glass microslides and polypropylene film has been discussed. [ABSTRACT FROM AUTHOR]

A number of hetero- and homobifunctional reagents have been reported to immobilize biomolecules on a variety of supports. However, efforts are on to search for a method, which is relatively simple, involving minimum of steps, cost effective, easy to reproduce, and that produces stable oligonucleotide arrays. Two new reagents, viz., [N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)- triethoxysilylpropyl-3-amine], and [N-(3-trifluoroethanesulfonyloxypropyl)anthraquinone-2- carboxamide] have been designed considering the above points. These reagents contain different functional groups at their two ends. In [N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)- triethoxysilylpropyl-3-amine], one end (triethoxysilyl) is capable of binding to the virgin glass surface and the other one consists of trifluoroethanesulfonate (tresyl) function specific toward aminoalkyl and mercaptoalkyl functionalities, which are easy to introduce at the 3′- or 5′-end of oligonucleotides. Likewise, in [N-(3-trifluoroethanesulfonyloxypropyl)anthraquinone-2- carboxamide], one end consists of photoactivatable moiety (anthraquinone) capable of reacting to a C-H containing surface and the tresyl function at the other end reacts specifically with aminoalkyl and mercaptoalkyl functionalities in modified oligonucleotides. These reagents have successfully been utilized to construct a number of oligonucleotide arrays and subsequently used for the detection of mismatches. [ABSTRACT FROM AUTHOR]

Array-based comparative genomic hybridization (array CGH) is becoming a prominent genomic technology with many important applications in biomedical research. Although several platforms of this technology have been published, successful implementation of this technology still demands technical expertise. Here, we describe the technology that has been developed and improved in the past few years are described. Our array CGH technology is primarily based on robust and readily implemented array production method. We also developed related protocols for using our bacterial artificial chromosomes CGH microarrays. This technology was successfully used to detect DNA copy-number alterations in various mouse and human samples. [ABSTRACT FROM AUTHOR]

Affinity mass spectrometry is a proteomics approach for selectively isolating target proteins from complex biological fluids for mass spectrometric analysis. When executed in high throughput mode through affinity pipets, the resulting volumetric mass spectrometry arrays enable rapid protein assaying from hundreds of samples. Furthermore, in combination with postcapture proteolytic degradation, this top-down proteomics approach can reveal structural features (i.e., modifications) in the protein sequences that are result of posttranslational modifications and/or point mutations. Described here in greater detail are the individual steps of the high throughput combination of affinity protein capture in antibody-derivatized affinity pipets, protein elution, and protein processing through enzyme-derivatized mass spectrometry targets. [ABSTRACT FROM AUTHOR]

Microarray technology, like many other surface-capture diagnostic methods, relies on fidelity of affinity interactions between a surface-bound probe (e.g., nucleic acid or antibody) and its target in the sample milieu to produce an assay signal specific to analyte. These interfacial interactions produce the assay result with the associated assay requirements for sensitivity, specificity, reproducibility, and ease-of-use. For surface-capture assays, surface properties play a critical role in this performance. Microarray surfaces are routinely immersed into aqueous target solutions of varying complexity, from simple saline or buffer solutions to serum, tissue, food, or microbiological lysates involving thousands of different solutes. The surface chemistry must not only be capable of immobilizing probes at high density in microscale patterned spots, retaining probe affinity for target within these spots, reducing target capture outside of these spots, but also be efficient at eliminating nontarget capture any where else on the surface. Historically, the development of surface chemistry with these specific "nonfouling" properties has been an intense interest for bioassays, with many types of architectures, molecular compositions, and performance capabilities across many different surface-capture assays. The unique environment of the bioassay, including the long-standing problems associated with high concentrations of "nontarget" proteins and other surface-active biomolecules in the assay milieu, has proven to be quite challenging to surface chemistry performance. Microarray technology designs with microspotted patterns must address these problems in these challenging dimensions in order to improve signal:noise ratios for captured target signals on surfaces. This chapter reviews principles of protein-surface interfacial physical chemistry, protein adsorption as a source of assay noise, and various approaches to control this interface in the context of surface-capture assay fabrication andimproving assay performance from complex milieu. Practical methods to modify surfaces for biological assay are presented. Polymer substrate coating methods, including "grafting from" and "grafting to" strategies, polymer brushes, and alternative surface modification methods are reviewed. Methods to assess biological "fouling" in the bioassay format are also discussed. [ABSTRACT FROM AUTHOR]

In almost all microarray technologies that are currently used, some type of surface chemistry serves as the interface between immobilized biomolecules and the solid support. Factors such as probe loading, spot morphology, and signal-to-noise ratio are all intimately linked to surface chemistry. Surface chemistry also significantly impacts important performance parameters such as three-dimensional structure of the immobilized biomolecules and nonspecific assay backgrounds. Here, an overview of the major types of surface chemistries currently used in printed microarrays is provided, with an emphasis on standard glass slide formats. The first part of this chapter focuses on DNA array surface chemistries, including both commercially fabricated and custom-made arrays. The second part of the chapter focuses on emerging protein, peptide, and carbohydrate array techniques. The intent is to provide the molecular biology researcher and bioanalytical or diagnostic specialist with a guide to the surface chemistry state-of-the-art for established and emerging array technologies. [ABSTRACT FROM AUTHOR]

The last decade has witnessed an impressive upsurge in the utilization of microarray platforms for biomedical research. However, the application of this emerging technology in medical practice lagged behind. This lag is understandable because there are specific issues pertaining to the utilization of clinical samples, which has to be rigorously addressed and overcome before microarrays enter mainstream medical practice. Such issues include cost, ethics, the complexity and heterogeneity of human tissue architecture, and their corresponding diseases, the type of tissues to be used, nucleic acids amplification, and experimental variability. As microarrays enter, albeit cautiously, the frontline of clinical practice, investigators and clinicians require to set up protocols that address these issues. This chapter decribes the methods used for nucleic acids preparation from frozen and formalin-fixed paraffin-embedded human tissues using macro-and microdissection and show their suitability for use in microarray experiments. [ABSTRACT FROM AUTHOR]

Microarray technology has its roots in high-throughput parallel synthesis of biomacromolecules, combined with combinatorial science. In principle, the preparation of arrays can be performed either by in situ synthesis of biomacromolecules on solid substrates or by spotting of ex situ synthesized biomacromolecules onto the substrate surface. The application of microarrays includes spatial addressing with target (macro) molecules and screening for interactions between immobilized probe and target. The screening is simplified by the microarray format, which features a known structure of every immobilized library element. The area of nucleic acid arrays is best developed, because such arrays are allowed to follow the biosynthetic pathway from genes to proteins, and because nucleic acid hybridization is a most straightforward screening tool. Applications to genomics, transcriptomics, proteomics, and glycomics are currently in the foreground of interest; in this postgenomic phase they are allowed to gain new insights into the molecular basis of cellular processes and the development of disease. [ABSTRACT FROM AUTHOR]

Gene expression microarrays are becoming increasingly widespread, especially as a way to rapidly identify putative functions of unknown genes. Accurate microarray data analysis, however, still remains a challenge. The recent availability of multiple types of high-throughput functional genomic data can facilitate accurate and effective analysis of microarray experiments and thereby accelerate functional annotation of sequenced genomes. But genomic data often sacrifice specificity for scale, yielding very large quantities of relatively lower quality data than traditional experimental methods. Advanced analysis methods are thus necessary to make accurate functional interpretation of these large-scale datasets. This chapter outlines recently developed methods that integrate the analysis of microarray data with sequence, interaction, localization, and literature data and further outlines specific problems in currently available integrated analysis technologies. [ABSTRACT FROM AUTHOR]

A method for systematically selecting the large number of sequences needed to custom design an oligonucleotide microarray was presented. This approach uses a Perl script to query sequence databases with gene lists obtained from previously designed (and publicly available) microarrays. Homologous sequences passing a user-defined threshold are returned and stored in a candidate gene database. Using this versatile technique, microarrays can be designed for any organism having sequence data. In addition, the ability to select specific input gene lists allows the design of microarrays tailored to address questions pertaining to specific pathways or processes. Given recent concerns about the accuracy of annotation in public sequence databases, it is also necessary to confirm the correct orientation of candidate sequences. This step is performed by a second Perl script that extracts protein similarity information from individual Unigene records, checks for consistency of features, and adds this information to the candidate gene database. Discrepancies between the orientations determined using protein similarities and that predicted by a given sequence's assigned orientation are readily apparent by querying the candidate gene database. [ABSTRACT FROM AUTHOR]

The human genome project has opened up a new page in scientific history. To this end, a variety of techniques such as microarray has evolved to monitor the transcript abundance for all of the organism's genes rapidly and efficiently. Behind the massive numbers produced by these techniques, which amount to hundreds of data points for thousands or tens of thousands of genes, there hides an immense amount of biological information. The importance of microarray data analysis lies in presenting functional annotations and classifications. The process of the functional classifications is conducted as follows. The first step is to cluster gene expression data. Cluster 3.0 and Java Treeview are widely used open-source programs to group together genes with similar pattern of expressions, and to provide a computational and graphical environment for analyzing data from DNA microarray experiments, or other genomic datasets. Clustered genes can later be decoded by Bulk Gene Searching Systems in Java (BGSSJ). BGSSJ is an XML-based Java application that systemizes lists of interesting genes and proteins for biological interpretation in the context of the gene ontology. Gene ontology gathers information for molecular function, biological processes, and cellular components with a number of different organisms. In this chapter, in terms of how to use Cluster 3.0 and Java Treeview for microarray data clustering, and BGSSJ for functional classification are explained in detail. [ABSTRACT FROM AUTHOR]

DNA duplex stability on oligonucleotide microarray was calculated using recently developed electrostatic theory of on-array hybridization thermodynamics. In this method, the first step is to finding the enthalpy and entropy of duplex formation in solution. This standard calculation was done with nearest-neighbor scheme and on-line software. Next the defined parameters and the array's single characteristic, the surface density of probes, are used to predict on-array duplex melting behavior. Reasonable accords of calculated and experimental melting curves for in situ synthesized microfluidic array were observed. The proposed method could be useful in microarray design and hybridization optimization. However, lack of melting curve measurements for different microarray platforms makes more experiments desirable to determine the method's accuracy. [ABSTRACT FROM AUTHOR]

This chapter outlines a typical workflow for micraorray data analysis. It aims at explaining the background of the methods as this is necessary for deciding upon a specific numerical method to use and for understanding and interpreting the outcomes of the analyses. We focus on error handling, various steps during preprocessing (clipping, imputing missing values, normalization, and transformation of data), statistic tests for variable selection and the use of multiple hypothesis testing procedures, various metrics and clustering algorithms for hierarchical clustering, principles, and results from principal components analysis and discriminant analysis, partitioning, selforganizing map, K-nearest neighbor classifier, and the use of a neural network and a support vector machine for classification. [ABSTRACT FROM AUTHOR]

We describe in this chapter the use of oligonucleotide or peptide microarrays (arrays) based on microfluidic chips. Specifically, three major applications are presented: (1) microRNA/small RNA detection using a microRNA detection chip, (2) protein binding and function analysis using epitope, kinase substrate, or phosphopeptide chips, and (3) protein-binding analysis using oligonucleotide chips. These diverse categories of customizable arrays are based on the same biochip platform featuring a significant amount of flexibility in the sequence design to suit a wide range of research needs. The protocols of the array applications play a critical role in obtaining high quality and reliable results. Given the comprehensive and complex nature of the array experiments, the details presented in this chapter is intended merely as a useful information source of reference or a starting point for many researchers who are interested in genomeor proteome-scale studies of proteins and nucleic acids and their interactions. [ABSTRACT FROM AUTHOR]

The development and characterization of protein microarrays fabricated on nanoengineered three-dimensional polyelectrolyte thin films (PET) deposited on glass slide by consecutive adsorption of polyelectrolytes in solutions through self-assembly process were described. Protein antibodies or antigens were immobilized in the PET-coated glass slides by electrostatic adsorption and entrapment of the porous structure of the three-dimensional polymer film and thus, establishing a platform for parallel analysis. A method for fabrication of cytokines antibodybased protein microarray for simultaneous detection of multiple cytokines on the PET-coated slides was described. Cytokines play an important role in a wide range of physiological process, such as innate immunity, apoptosis, angiogenesis, cell growth, and differentiation. Therefore, simultaneous measurement of multiple cytokine expression levels is vital to reveal the complex cytokine network and to understand the development of certain human diseases. The protein microarray was printed by robotically spotting nine human cytokine and growth factor capture antibodies onto planar glass substrates. The fluoroimmunoassay of printed cytokine antibody microarrays were performed by incubating with cytokine samples, then binding by biotin-conjugated detection antibodies, and detecting by fluorophore conjugated streptavidin. This sandwich immunoassay-based protein microarrays protocol was developed for detection of multiple expression levels simultaneously with commercial available biotin-labeled detection antibody, so that no labeling of sera samples in required. This method was also optimized specifically for the special requirements of the cytokine detection, with special attention paid to selecting the surface chemistry of array substrate, array printing buffer and blocking buffer, and the fluorescent detection settings that yielded the highest sensitivity and selectivity against the lowest background. The dynamic ranges of the parallel assay for cytokines were aroundtwo to three orders of magnitude with limit of detection <10 pg/mL. This cytokine detection protein microarray system can be extended to a larger menu of cytokines and growth factors for applications such as profiling of cytokine expression. [ABSTRACT FROM AUTHOR]

Small molecule detection poses special problems during analysis whether hormones in a clinical setting or pesticides from environmental monitoring. Traditional analysis involves procedures like high-pressure liquid chromatography, gas chromatography, or mass spectrometry, or a combination of the three. Microarray procedures have recently evolved into a technique capable of replacing many of these assays, utilizing the strong and specific binding of a binder (e.g., an antibody) to a given target, even in a quantitative manner. A higher sensitivity can be obtained using microarrays were shown, even without the concentration of the sample beforehand. The sensitivity is high enough for monitoring most clinically relevant markers and current regulatory pesticide levels. The microarray technique has additionally parallelism in sample analysis. The same sample can be analyzed for many targets at the same time, and under the same conditions. In the present protocol pesticide detection by microarray analysis is presented. [ABSTRACT FROM AUTHOR]

Small molecules, such as metabolites and hormones, interact with proteins to regulate numerous biological pathways, which are often aberrant in disease. Small molecule drugs have been successfully exploited to specifically perturb such processes and thereby, decrease and even eliminate disease progression. Although there are compelling reasons to fully characterize interactions of small molecules with all proteins from an organism for which an intended drug regimen is planned, currently available technologies are not yet up to this task. High-content functional protein microarrays, containing hundreds to thousands of proteins, are new tools that show great potential for meeting this need. In this chapter, we review examples and methods for profiling small molecules on high-content functional protein arrays and discuss considerations for troubleshooting. [ABSTRACT FROM AUTHOR]

Tumor-stroma interactions are of great importance not only for the development and progression of primary prostate carcinoma but probably also for the establishment of metastasis. Fibroblasts are an important stromal cell type encountered by metastatic tumor cells at different sites. In previous investigations, we had found that media conditioned by three metastatic prostate cancer cell lines (LNCaP, PC-3, and DU-145) induced cultured nonprostatic fibroblasts to proliferate or to express matrix-metalloproteinase-1 considered important for tumor invasion. Fibroblast-conditioned media in turn stimulate proliferation of DU-145 cells and migration of PC-3 cells. Both tumor cells and fibroblasts secrete VEGF suggesting that not only metastatic but also stromal cells at metastatic sites contribute to the vascularization of metastasis necessary for continuous growth. In order to better understand the reciprocal tumor-stroma cross-talk in molecular terms we used the mRNA extracted from stimulated and unstimulated neoplastic and fibroblastic stromal cells for cDNA array hybridization using Affymetrix® chips. The three prostate cell lines influenced the fibroblasts nearly in the same manner. In particular proteins involved in cell adhesion, cell-cell contact, and cell cycle regulation were downregulated in stimulated fibroblasts. In contrast, fibroblasts affected every prostate cancer cell line in different ways, which may be because of the different origin of the metastatic prostate cancer cell lines. [ABSTRACT FROM AUTHOR]

The ability to perform microarray-based immunoassays without the need for wells or other fluid barriers were demonstrated. Both contact and noncontact microarray printing technology is used to prepare spotted arrays of analyte binding sites, as well as, to deliver samples, secondary antibodies and other signal development reagents directly to these sites in a parallel fashion are called as overprint immunoassays. A micro-ELISA is demonstrated based upon the use of Protein A as a universal microarray. All components of the assay (capture antibody, antigen, and signal development reagents) were site-specifically dispensed in parallel fashion to the surface in nanoliter volumes. This represents a 1000-fold reduction in reagent consumption from that used in a conventional 96-well microtiter plate assay. Overprinting nanoliter volumes directly onto 200-300 µm spots yields similar levels of sensitivity achieved with the bulk dispensing of microliter volumes into wells. [ABSTRACT FROM AUTHOR]

Automated target preparation for gene expression analysis eliminates the time-consuming and labor-intensive manual process, which is error prone and diverts scientists from value added activities. Target preparation methods were developed, on the fully integrated ArrayPlex SA® system, based on the field proven Biomek FX®, which streamlined the target preparation procedure allowing up to 96 samples to be processed in less than 36 h. The process is comprised of three functional methods, cDNA synthesis, in vitro transcription, and fragmentation, providing the users with the ability to consolidate runs for optimal use of instrument time and minimize reagent cost. Starting with sets of eight tRNA samples, the cDNA synthesis method synthesizes the first and second strand DNA followed by a cDNA clean-up step using an ultrafiltration plate. The in vitro transcription method then amplifies and biotin-labels the cDNA to cRNA in 6 h at 37°C, and purifies the product using a solid support extraction plate. Finally, the fragmentation method quantifies the cRNA, adjusts the concentration to the recommended 0.625 µg/mL and fragments the cRNA prior to an off-line hybridization. Universal human reference RNA with concentration ranging from 1 to 7.5 µg were prepared on the ArrayPlex SA, compared against a manual method and scanned using Affymetrix human genome U133 Plus 2.0 array GeneChip® cartridges. Nested analysis of variance was then performed to identify sources of variability between the automated and manual methods. [ABSTRACT FROM AUTHOR]

A large number of human genetic diseases, bacterial drug resistances, and single-nucleotide polymorphisms are caused by gene mutations. Rapid and high-throughput mutation detection methods are urgently demanded. A protein chip method for detection of single-base mismatches and unpaired bases of DNA was developed using a genetic fusion molecular system Trx-His6-(Ser-Gly)6-Strep tagII-(Ser-Gly)6-MutS (THLSLM). The THLSLM coding sequence was constructed by attaching Strep tag II and mutS gene to the vector pET32a (+) sequentially with insertion of a (Ser-Gly)6 coding sequence before and behind Strep tagII gene, respectively. The fusion protein THLSLM was expressed in Escherichia coli AD494 (DE3) and purified using Ni2+-chelation affinity resin. The results of bioactivity assay showed that THLSLM both binds to mismatched DNA and interacts with streptavidin. THLSLM was immobilized on the chip matrix coated with the streptavidin through Strep tagII-streptavidin binding reaction. The resulting protein chip was used to detect the mismatched and unpaired mutations in the synthesized oligonucleotides, as well as a single-base mutation in rpoB gene from Mycobacterium tuberculosis, with high specificity. The method could potentially serve as a platform to develop the high-throughput technology for screening and analysis of genetic mutations. [ABSTRACT FROM AUTHOR]

Although the majority of microarray studies have been directed toward RNA expression profiling (functional genomics) and increasingly toward proteomics, a steady increase in the use of microarrays as platforms for DNA genotyping has occurred over the past 5 yr. Multiple array-based chemistries have been developed in order to genotype single nucleotide polymorphisms. Conceptually, the simplest of these microarray genotyping technologies is based on the dideoxynucleotide chemistry of mini-sequencing by arrayed primer extension, whereby oligonucleotide probes (preprinted on the array) are extended by a single nucleotide base. This enzymecatalyzed single base extension reaction is dependent on the sequence (genotype) of the template nucleic acid (sample) that is temporarily hybridized to the probes. Utilization of all four dideoxynucleotides, each conjugated to a different fluorophore, allows genotyping by spectral differentiation of the single base extension reaction products. [ABSTRACT FROM AUTHOR]

With the completion of the Human Genome Project, the microarray technology has evolved into a sophisticated platform by which complex diseases such as cancer, can be studied at the genome, transcriptome, and proteome levels. Here, various microarray platforms, namely comparative genomic hybridization, cDNA, oligonucleotide, and protein-based microarrays are exploited to study genomic copy-number changes in a human cancer cell line and correlate these genomic aberrations with their expression at mRNA and protein levels. The protocols described therein can be assimilated for the study of other human tissues including cancerous ones. [ABSTRACT FROM AUTHOR]

A quantitative screening method was developed to evaluate the quality of cDNA libraries constructed by suppression subtraction hybridization (SSH) or other enrichment techniques. The SSH technique was adapted to facilitate screening of the resultant library on a small number of glass slide microarrays. A simple data analysis pipeline named SSHscreen using "linear models for microarray data" (limma) functions in the R computing environment was developed to identify clones in the cDNA libraries that are significantly differentially expressed, and to determine if they were rare or abundant in the original treated sample. This approach facilitates the choice of clones from the cDNA library for further analysis, such as DNA sequencing, Northern blotting, RT-PCR, or detailed expression profiling using a custom cDNA microarray. Furthermore, this strategy is particularly useful for studies of nonmodel organisms for which there is little genome sequence information. [ABSTRACT FROM AUTHOR]

Microarrays are a powerful laboratory tool for the simultaneous assessment of the activity of thousands genes. Remarkable advances in biological sample collection, preparation and automation of hybridization have enabled the application of microarray technology to large, population-based studies. Now, microarrays have the potential to serve as screening tools for the detection of altered gene expression activity that might contribute to diseases in human populations. Reproducible and reliable microarray results depend on multiple factors. In this chapter, biological sample parameters are introduced that should be considered for any microarray experiment. Then, the microarray technology that we have successfully applied to limited biological sample from all our molecular epidemiology studies is detailed. This reproducible and reliable approach for using microarrays should be applicable to any biological questions asked. [ABSTRACT FROM AUTHOR]

The microRNAs (or miRNAs) are small noncoding RNAs (21—25 nt) that are processed from large hairpin RNA precursors and are believed to be involved in a wide range of developmental and cellular processes, by either repressing translation or triggering mRNA interference (RNA interference). Over 200 of distinct genes encoding miRNAs have been identified through either computer-assisted approaches or complementary deoxyribonucleic acid cloning strategies in many organisms including worm, plants, flies, mouse, and human. Recently, a microarray based robust method to profile miRNAs expression in organs cell lines and tissues in mammalians were developed (1). Using this method, we have identified a group of miRNAs preferentially expressed in human primary adipocytes and knocking down one such miRNA (miRNA 143) reverses the differentiation process (2). Groups of kidney specific miRNAs share evolutionary conserved phylogenetic foot print Ets1 in the upstream of the miRNA, possibly important for kidney physiological maintenance were reported. A detail protocol of the method are discussed to develop miRNA profile for global gene expression in tissues, organs, and cell lines in eukaryotes. [ABSTRACT FROM AUTHOR]

A move away from high-density screening array formats and the implementation of probes specifically identifying targets for application in low-density hybridization capture analyses is growing in importance. Some of the highest specificity for bioassays that encompasses use of hybridization has enlisted hydrolysis probes in real-time PCR. It is demonstrated that by employing 5′-end-tethering to glass of hydrolysis PCR probe sequences that these can be employed as capture probe sequences. The probes retain hybridization binding specificity to their PCR amplicons and specificity of hybridization was achieved across all probes at equivalent stringency. This suggests that probes identified for use in hydrolysis PCR using one set of temperature cycling parameter can also be applied to a microarray, where again the probes all exhibit similar specificity of interact at analogous hybridization conditions. The procedure permits the exact same PCR amplicon sequences to be used in both quantitative PCR and qualitative microarray assay formats. [ABSTRACT FROM AUTHOR]

Based on the selection of a suitable surface chemistry and bearing the option for optimization using a defined workflow, standard experimental protocols for the processing of microarrays can be used as starting points for a successful experiment. In Chapters 2 and 3, general quality considerations and the selection of surface chemistry have been discussed. A workflow for the selection of slide surface architectures and the optimization of microarray processing parameters also has been described. In the present article, processing parameters for DNA, peptide, antibody, and carbohydrate microarrays are outlined that serve as a first recommended step in the iterative establishment process. For a number of popular applications of microarray technology the outlined protocols can be applied to directly generate highquality results. [ABSTRACT FROM AUTHOR]

As the performance of microarray experiments is directly dependent on the quality of the materials, the suitability of the protocols, and the accuracy of the work performed, optimization of existing microarray workflows is needed in almost every experiment to achieve higher quality and meaningfulness of the generated data. In the following, we describe a workflow for the optimization of microarray processing parameters, based on the previously selected surface structure. Four simple model experiments with dye-labeled compounds is used to determine crucial experimental parameters including spotting concentration, spotting solution, immobilization efficiency, and blocking conditions even in cases where recommendations from the slide manufacturer or from the literature are missing. In this article, processing parameters for DNA, peptide, antibody, and carbohydrate microarrays are outlined. The applicability of the model experiments is demonstrated and described in detail on the example of short oligonucleotides. [ABSTRACT FROM AUTHOR]

The complexity of workflows for the production of high quality microarrays asks for the careful evaluation and implementation of materials and methods. As a cornerstone of the whole microarray process, the microarray substrate has to be chosen appropriately and a number of crucial considerations in respect to matching the research question with the technical requirements and possibilities have to be taken into account. In the following, how to lay the fundamental for high performance microarray experiments by evaluating basic quality requirements and the selection of suitable slide surface architectures for a variety of applications was concentrated. [ABSTRACT FROM AUTHOR]

Among vertebrates, the mammalian systems that are frequently used to investigate questions related to human health have gained the most benefit from microarray technology to date. However, it is clear that biological investigations and the generalized conclusions drawn from them, can only be enhanced by including organisms in which specific processes can be readily studied because of their genetic, physiological, or developmental disposition. As a result, the field of functional genomics has recently begun to embrace a number of other vertebrate species. This review summarizes the current state of microarray technology in a subset of these vertebrate organisms, including Xenopus, Rana, zebrafish, killifish (Fundulus sp.), medaka (Oryzias latipes), Atlantic salmon, and rainbow trout. A summary of various applications of microarray technology and a brief introduction to the steps involved in carrying out a microarray experiment are also presented. [ABSTRACT FROM AUTHOR]