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This brief presents a 12-bit ultra-low-power asynchronous successive approximation register (SAR) analog-to-digital converter (ADC). A voltage-controlled delay line (VCDL) based open-loop time-domain comparator is proposed and analyzed, achieving low noise and ultra-low power performance. By employing the mixed switching scheme, the segmented capacitive digital-to-analog converter (CDAC) arrays as well as the synchronous data-weighted averaging (DWA) calibration block, the proposed SAR ADC can operate from 1.8 V down to 0.8 V at 20-200 kS/s. The designed ADC is fabricated in a 0.18-um CMOS process and the measurement results show the proposed SAR ADC achieves an SNDR of 65-dB with power consumption of 647 nW from a 0.8 V power supply at 20 kS/s.

Because the strategy of stopping bus lines during an epidemic can negatively impact residents, this study proposes a bus passenger flow control model to optimize the safety of and access to bus transport. The information interaction environment can provide a means for the two-way regulation of buses and passengers. In this model, passengers first request their pick-up and drop-off location, and then the bus feeds back information on whether it accepts the request. Through this method, passenger flow control can be realized through complete information interaction. The study aimed to establish a multi-objective function that minimizes the weighted total cost of the safety cost, the passenger travel cost, and the bus travel cost during an epidemic. The constraints were the full load and riding rates of urban buses in peak periods under the condition of epidemic prevention and control. The results showed that, in the morning peak period, the passenger flow control scheme reduced the passenger infection probability by 17.89%, compared with no passenger flow control scheme. The weighted total cost of the epidemic safety cost, the passenger travel cost, and the bus operation cost was reduced by 8.04%. The optimization effect of the passenger flow control scheme of this model is good, and not only reduces the probability of passengers being infected, but also meets the requirements of epidemic prevention and the travel needs of residents.

Environment-friendly high color purity blue zinc selenide (ZnSe) quantum dot-based light-emitting diodes (QLEDs) are promising candidates in next-generation display applications. However, due to the large bandgap of ZnSe (2.7 eV), the reported electroluminescence (EL) wavelengths of ZnSe QLEDs are mainly located within the range from purple to violet blue, and preparing blue emitting (>445 nm) ZnSe QLEDs remains challenging. Herein, we report a seed-mediated and double shell strategy to synthesize large-sized blue ZnSe/ZnS/ZnS core/shell/shell quantum dots (QDs). The as-prepared QDs possess excellent features including narrow full widths at half-maximum (11-19 nm), tunable emission wavelengths (410-451 nm), and high photoluminescence quantum yields (≥50%). Using ZnSe/ZnS/ZnS QDs as emitters in an inverted device, a color saturated blue QLED with an EL wavelength of 446 nm, a maximum luminance of 106 cd m-2, a current efficiency of 0.94 cd A-1, and an EQE of 2.62% is successfully fabricated. These results indicate that blue ZnSe QLEDs have great potential for future display technologies.

All-solution processed inverted quantum dot light-emitting diodes (QLEDs) are promising candidates for lighting and display applications. While the external quantum efficiency of inverted devices is comparable to that of devices with normal structure, achieving both high efficiency and long lifetime in such inverted devices remains challenging. Herein, we report an all-solution processed inverted green QLED capable of achieving a 96.42 cd A−1 current efficiency, a 25.04% external quantum efficiency, and a 4943.6 h half-lifetime. This external quantum efficiency exceeds reported literature values, and the lifetime is over 19 times greater than those of previously reported all-solution processed inverted QLEDs. Such excellent performance is attributed to the precisely controlled double ZnS shells of CdSeZnS/ZnS/ZnS quantum dots, which can effectively suppress Auger recombination and Forster resonance energy transfer, as well as decrease efficiency roll-off at high driving currents. Overall, this study suggests that shell engineering of quantum dots may provide an effective means of accelerating the inverted QLEDs to meet requirements for practical display and lighting applications.

Nonredox metal ions have been widely recognized to be important in a wide range of biological and chemical oxidations as Lewis acids (LA). However, the role of LA in peroxymonosulfate (PMS) activation for wastewater treatment has not been considered until now. This study shows that oxidizing power of PMS can be promoted after binding nonredox metal ions such as Ca

Nonredox metal ions have been widely recognized to be important in a wide range of biological and chemical oxidations as Lewis acids (LA). However, the role of LA in peroxymonosulfate (PMS) activation for wastewater treatment has not been considered until now. This study shows that oxidizing power of PMS can be promoted after binding nonredox metal ions such as Ca2+ as LA, leading to the easier reduction of the oxidant to radicals and substantial enhancement of dye degradation by employing manganese oxides OMS-2 as model catalysts. Increased with Lewis acidity of the metal ion, the rate of PMS decomposition enhanced linearly, while the dye degradation rate first increased and then declined due to the formation of a larger amount of dioxygen. The interactions between Ca2+ and PMS were further investigated by Raman, cyclic voltammetry and XPS; and the detailed mechanism of PMS activation was proposed. The performance of Ca2++OMS-2/PMS system under different conditions was also studied. The findings indicate the importance of LA in PMS activation reaction and their role must be considered in other transition metal oxides/PMS systems. It will be also helpful to design new and highly active catalysts for the reactions.

Structurally defected cryptomelane-type octahedral molecular sieve (SDOMS-2) was successfully prepared at room temperature for efficient activation of peroxymonosulfate (PMS). The catalyst was easily fabricated by redox reaction of acidic KMnO4 and MnSO4 with the presence of graphite at 25 °C for 96 h. XRD and N2 adsorption-desorption measurements confirmed the OMS-2 crystal with a high surface area of 118 m2/g. Evidence by Raman, XPS, cyclic voltammogram (CV), SEM and TEM, the slow-growing SDOMS-2 nanorods exhibited more structural defects and lower valance of manganese than the conventional OMS-2 prepared by a refluxing method. The SDOMS-2 catalyst showed high activity and stability in terms of Acid Orange 7 (AO7) degradation in the presence of PMS, and the initial degradation rate was 4 times of the refluxed OMS-2. Synthesis conditions influenced the surface morphology and detective structure of OMS-2, and thus appeared to play critical roles in PMS activation. Based on the scavenger experiments, 1O2 was identified as dominant oxygen species in the SDOMS-2 + PMS system for AO7 degradation. This study demonstrates a novel and simple technology for the preparation of SDOMS-2 for PMS activation.

We report a rational strategy to design and construct multiple small perturbation mutagenesis (SPM) libraries using massively parallel synthesis of oligonucleotides on a microchip for affinity maturation of an engineered anti-ErbB2 antibody chA21. On the basis of a comprehensive analysis of the sequence and structural relationships of six complementary determination regions (CDRs) in the Kabatman database, a computational algorithm was developed to introduce single-site and double-site mutations into variable CDR positions using ambiguous nucleotides. The six SPM libraries were composed of 419 degenerate oligonucleotides that can be expanded into 161,832 unique CDR sequences with a high coverage ratio of 95% natural amino acid diversity. We used Illumina next-generation sequencing to demonstrate that the synthetic CDR library sequences, as well as relative quantities per sequence, can be controlled precisely by adjusting reaction chamber assignment and input nucleoside composition. The microchip-synthesized oligonucleotides were used for construction of single-chain antibody fragment (scFv) phage libraries through one-step mutagenic PCR of double-stranded plasmids with >10 6 E. coli transformants. A variant with combinatorial mutations from four individual CDRs achieved more than 19-fold affinity increase. The strategy described herein should be broadly applicable to affinity and selectivity studies of antibodies and other proteins.

Breast milk is a complex liquid rich in immunological components that affect the development of the infant's immune system. Exosomes are membranous vesicles of endocytic origin that are found in various body fluids and that can mediate intercellular communication. MicroRNAs (miRNAs), a well-defined group of non-coding small RNAs, are packaged inside exosomes in human breast milk. Here, we identified 602 unique miRNAs originating from 452 miRNA precursors (pre-miRNAs) in human breast milk exosomes using deep sequencing technology. We found that, out of 87 well-characterized immune-related pre-miRNAs, 59 (67.82%) are presented and enriched in breast milk exosomes (P10(-16), χ(2) test). In addition, compared with exogenous synthetic miRNAs, these endogenous immune-related miRNAs are more resistant to relatively harsh conditions. It is, therefore, tempting to speculate that these exosomal miRNAs are transferred from the mother's milk to the infant via the digestive tract, and that they play a critical role in the development of the infant immune system.

Breast milk is a complex liquid rich in immunological components that affect the development of the infant's immune system. Exosomes are membranous vesicles of endocytic origin that are found in various body fluids and that can mediate intercellular communication. MicroRNAs (miRNAs), a well-defined group of non-coding small RNAs, are packaged inside exosomes in human breast milk. Here, we identified 602 unique miRNAs originating from 452 miRNA precursors (pre-miRNAs) in human breast milk exosomes using deep sequencing technology. We found that, out of 87 well-characterized immune-related pre-miRNAs, 59 (67.82%) are presented and enriched in breast milk exosomes (P < 10-16, χ2 test). In addition, compared with exogenous synthetic miRNAs, these endogenous immune-related miRNAs are more resistant to relatively harsh conditions. It is, therefore, tempting to speculate that these exosomal miRNAs are transferred from the mother's milk to the infant via the digestive tract, and that they play a critical role in the development of the infant immune system.

A large number of plant microRNAs (miRNAs) are now documented in the miRBase, among which only 30 are for Solanum lycopersicum (tomato). Clearly, there is a far-reaching need to identify and profile the expression of miRNAs in this important crop under various physiological and pathological conditions. In this study, we used an in situ synthesized custom microarray of plant miRNAs to examine the expression and temporal presence of miRNAs in the leaves of tomato plants infected with Cucumber mosaic virus (CMV). Following computational sequence homology search and hairpin structure prediction, we identified three novel tomato miRNA precursor genes. Our results also show that, in accordance with the phenotype of the developing leaves, the tomato miRNAs are differentially expressed at different stages of plant development and that CMV infection can induce or suppress the expression of miRNAs as well as up-regulate some star miRNAs (miRNA*s) which are normally present at much lower levels. The results indicate that developmental anomalies elicited by virus infection may be caused by more complex biological processes.

Background: MicroRNAs (miRNAs), a large family of short endogenous RNAs known to post-transcriptionally repress gene expression, participate in the regulation of almost every cellular process. Changes in miRNA expression are associated with many pathologies. Ovarian folliculogenesis and testicular spermatogenesis are complex and coordinated biological processes, in which tightly regulated expression and interaction of a multitude of genes could be regulated by these miRNAs. Identification and preliminary characterization of gonad-specific miRNAs would be a prerequisite for a thorough understanding of the role that miRNA-mediated posttranscriptional gene regulation plays in mammalian reproduction.Method: Here, we present the identification of a repertoire of porcine miRNAs in adult ovary and testis using deep sequencing technology. A bioinformatics pipeline was developed to distinguish authentic mature miRNA sequences from other classes of small RNAs represented in the sequencing data.Results: Using this approach, we detected 582 precursor hairpins (pre-miRNAs) encoding for 732 mature miRNAs, of which 673 are unique. Statistically, 224 unique miRNAs (out of 673, 33.28%) were identified which had significant differential expression (DE) between ovary and testis libraries (P < 0.001). Most of DE miRNAs located on the X chromosome (X-linked miRNAs) (24 out of 34, 70.59%) significantly up-regulated in ovary versus testis (P < 0.001). Predictably, X-linked miRNAs are expressed in a testis-preferential or testis-specific pattern. To explore the potential for co-expression among genomic location clusters of X-linked miRNAs, we surveyed the relationship between the distance separating miRNA loci and the coordinate expression patterns of 32 high confidence X-linked miRNAs in seven normal pig tissues using the real-time quantitative PCR (q-PCR) approach. Our results show that proximal pairs of miRNAs are generally co-expressed implying that miRNAs within 50 kb of genomic bases are typically derived from a common transcript.Conclusions: The present study characterizes the miRNA transcriptome of adult porcine gonads, with an emphasis on the co-expression patterns of X-linked miRNAs. Our report should facilitate studies of the organ-specific reproductive roles of miRNAs.

Phosphoprotein-binding domains (PPBDs) mediate many important cellular and molecular processes. Ten PPBDs have been known to exist in the human proteome, namely, 14-3-3, BRCT, C2, FHA, MH2, PBD, PTB, SH2, WD-40 and WW. PepCyber:P approximately PEP is a newly constructed database specialized in documenting human PPBD-containing proteins and PPBD-mediated interactions. Our motivation is to provide the research community with a rich information source emphasizing the reported, experimentally validated data for specific PPBD-PPEP interactions. This information is not only useful for designing, comparing and validating the relevant experiments, but it also serves as a knowledge-base for computationally constructing systems signaling pathways and networks. PepCyber:P approximately PEP is accessible through the URL, http://www.pepcyber.org/PPEP/. The current release of the database contains 7044 PPBD-mediated interactions involving 337 PPBD-containing proteins and 1123 substrate proteins.

Bruton's tyrosine kinase (Btk) and interleukin-2-inducible T cell kinase (Itk) are members of the TEC family of nonreceptor tyrosine kinases and are expressed primarily in B and T cells, respectively. Both kinases are critically involved in lymphocyte development and signal transduction. In particular, Btk and Itk regulate calcium mobilization subsequent to antigen receptor stimulation. Small molecule antagonists that specifically inhibit either Btk or Itk may allow for selective modulation of B cell or T cell activity and may be useful in treating inflammatory and autoimmune conditions. We have developed a medium-throughput fluorescent imaging plate reader (FLIPR)- based calcium flux assay that can be used to assay potential Btk and Itk inhibitors. This assay takes advantage of Btk-deficient DT40 (DT40-Btk-/-) chicken B cells, which are unable to mobilize calcium in response to cross-linking of their B cell receptor (BCR). Ectopic expression of TEC family kinases can restore antigen receptor signaling in these cells. We have generated stable DT40-Btk-/- lines expressing either wild-type human Btk (huBtk) or a chimeric Btk-Itk kinase (huBtk-Itk) molecule-a Btk protein whose kinase domain has been replaced by the kinase domain of Itk. Expression of either huBtk or huBtk-Itk in DT40-Btk-/- cells restores calcium flux in response to BCR engagement. Using Btk- and Itk-selective inhibitors, we show that inhibition of calcium responses in huBtk-Itk-DT40-Btk-/- cells and huBtk-DT40-Btk-/- cells is dependent on the Itk or Btk kinase domain, respectively. Thus, the FLIPR assay described here can be used to assess, compare, and rank the potency and selectivity of inhibitors of Itk and Btk kinases.

A homoditopic monomer containing two bis(m-phenylene)-32-crown-10 units and a complementary homoditopic monomer containing two paraquat moieties were designed and prepared. At high equimolar concentrations (>40 mM) they self-organize to form a linear supramolecular polymer in solution as confirmed by proton NMR spectroscopy, mass spectrometry, and viscosity studies.

A versatile microreactor platform featuring a novel chemical-resistant microvalve array has been developed using combined silicon/polymer micromachining and a special polymer membrane transfer process. The basic valve unit in the array has a typical 'transistor' structure and a PDMS/parylene double-layer valve membrane. A robust multiplexing algorithm is also proposed for individual addressing of a large array using a minimal number of signal inputs. The in-channel microvalve is leakproof upon pneumatic actuation. In open status it introduces small impedance to the fluidic flow, and allows a significantly larger dynamic range of flow rates (∼ml min −1 ) compared with most of the microvalves reported. Equivalent electronic circuits were established by modeling the microvalves as PMOS transistors and the fluidic channels as simple resistors to provide theoretical prediction of the device fluidic behavior. The presented microvalve/reactor array showed excellent chemical compatibility in the tests with several typical aggressive chemicals including those seriously degrading PDMS-based microfluidic devices. Combined with the multiplexing strategy, this versatile array platform can find a variety of lab-on-a-chip applications such as addressable multiplex biochemical synthesis/assays, and is particularly suitable for those requiring tough chemicals, large flow rates and/or high-throughput parallel processing. As an example, the device performance was examined through the addressed synthesis of 30-mer DNA oligonucleotides followed by sequence validation using on-chip hybridization. The results showed leakage-free valve array addressing and proper synthesis in target reactors, as well as uniform flow distribution and excellent regional reaction selectivity. (Some figures in this article are in colour only in the electronic version)

Testing the many hypotheses from genomics and systems biology experiments demands accurate and cost-effective gene and genome synthesis. Here we describe a microchip-based technology for multiplex gene synthesis. Pools of thousands of ‘construction’ oligonucleotides and tagged complementary ‘selection’ oligonucleotides are synthesized on photo-programmable microfluidic chips1, released, amplified and selected by hybridization to reduce synthesis errors ninefold. A one-step polymerase assembly multiplexing reaction assembles these into multiple genes. This technology enabled us to synthesize all 21 genes that encode the proteins of the Escherichia coli 30S ribosomal subunit, and to optimize their translation efficiency in vitro through alteration of codon bias. This is a significant step towards the synthesis of ribosomes in vitro and should have utility for synthetic biology in general.

Development of synthesis methods using high yield acid-labile rather than photolabile group protected monomers is desirable to produce microarrays of superior quality, general adaptability and reduced cost of genetic research. However, using solution photochemistry for acid-labile deprotection requires the reaction sites being isolated from each other to prevent diffusion of reagents during a photolytic reaction. We present fabrication methods of two types of microwell array chips with different isolation strategies for carrying out parallel oligonucleotide DNA (oDNA) synthesis in the liquid phase: electroplated microwell array chips with an isolation wall and a mechanical sealing, and etched patterned microwell array chips with surface tension isolation. Both surface and bulk micromachining technologies were used for fabrication of those chips. We also discuss various problems regarding fabrication and use. The validated fidelity from the single mismatch detection of oligonucleotide DNA microwell array chips is also given to prove successful isolation of the liquid during a photolytic reaction.

There is substantial interest in implementing a bioinformatics tool that allows the design of oligonucleotides to support the development of in vitro gene synthesis. Current protocols to make long synthetic DNA molecules rely on the in vitro assembly of a set of short oligonucleotides, either by ligase chain reaction (LCR) or by assembly PCR. Ideally, such oligonucleotides should represent both strands of the final DNA molecule. They should be adjacent on the same strand and overlap the complementary oligonucleotides from the second strand to ensure good hybridization during assembly. This implies that the thermodynamic properties of each oligonucleotide have to be consistent across the set. Furthermore, any given oligonucleotide has to be totally specific to its target to avoid the creation of incorrectly assembled sequences. We have developed Gene2Oligo (http://berry.engin.umich.edu/gene2oligo/), a web-based tool that divides a long input DNA sequence into a set of adjacent oligonucleotides representing both DNA strands. The length of the oligonucleotides is dynamically optimized to ensure both the specificity and the uniform melting temperatures necessary for in vitro gene synthesis. We have successfully designed and used a set of oligonucleotides to synthesize the Saccharomyces cerevisiae cytochrome b5 by using both LCR and assembly PCR.

The technologies enabling the creation of large scale, miniaturized peptide or protein microarrays are emerging. The focuses of this review are the synthesis and applications of peptide and peptidomimetic microarrays, especially the light directed parallel synthesis of individually addressable high density peptide microarrays using a novel photogenerated reagent chemistry and digital photolithography (Gao et al., 1998, J. Am. Chem. Soc. 120, 12698; Pellois et al. 2002, Nat. Biotechnol. 20, 922). Concepts related to the synthesis are discussed, such as the reactions of photogenerated acids in the deprotection step of peptide synthesis or oligonucleotide synthesis, and the applications of high density peptide chips in antibody binding assays are discussed. Peptide chips provide versatile tools for probing antigen-antibody, protein-protein, peptide-ligand interactions and are basic components for miniaturization, automation, and system integration in research and clinical diagnosis applications.

This contribution presents a brief overall look of the methods for the preparation of various types of DNA microarrays and a thorough examination of the methods for in situ synthesis of oligonucleotide microarrays.

Peptide and peptidomimetic molecule arrays are emerging powerful tools for parallel screening of binding in proteomics and pharmaceutical discovery research. Up to now the common method of preparing peptide arrays was based on spotting on glass using a library of presynthesized peptides. However, due to the large number of monomers (amino acids) it is not possible to have combinatorial libraries which include all combinations of natural and synthetic amino acids. We describe a very flexible on-chip oligopeptide synthesis method which uses the well developed t-Boc based solid state synthesis chemistry. A very high degree of flexibility is achieved by using light photo generated acids and maskless projection lithography for spatially directed deprotection. Use of microfluidic chips enables moderately high densities, short reaction times and off-the-shelf chemicals. Examples are given from synthesis of metal ion binding peptides and epitope binding assays.

Achieving high fidelity chemical synthesis on glass plates has become increasingly important, since glass plates are substrates widely used for miniaturized chemical and biochemical reactions and analyses. DNA chips can be directly prepared by synthesizing oligonucleotides on glass plates, but the characterization of these micro-syntheses has been limited by the sub-picomolar amount of material available. Most DNA chip syntheses have been assayed using in situ coupling of fluorescent molecules to the 5′-OH of the synthesized oligonucleotides. We herein report a systematic investigation of oligonucleotide synthesis on glass plates with the reactions carried out in an automated DNA synthesizer using standard phosphoramidite chemistry. The analyses were performed using 32P gel electrophoresis of the oligonucleotides cleaved from glass plates to provide product distribution profiles according to chain length of oligonucleotides. 5′-Methoxythymidine was used as the chain terminator, which permits assay of coupling reaction yields as a function of chain length growth. The results of this work reveal that a major cause of lower fidelity synthesis on glass plates is particularly inefficient reactions of the various reagents with functional groups close to glass plate surfaces. These problems cannot be detected by previous in situ fluorescence assays. The identification of this origin of low fidelity synthesis on glass plates should help to achieve improved synthesis for high quality oligonucleotide microarrays.

We report a detailed study of molecular beam epitaxial growth of ZnS films on bare and arsenic-passivated vicinal Si(100) surfaces. This study elucidates the initiation of microtwinning and stacking-fault defects on double-stepped substrate surfaces. The study also sheds light on the function of arsenic passivation in reducing crystal defects in ZnS epitaxial layers. Three substrate surfaces, Si(100) 2×1, Si(100):As 2×1, and Si(100):As 1×2, were used for the ZnS epitaxial growth studies. Adsorption experiments were performed to demonstrate the chemical passivation effect of an arsenic overlayer. Reflection high-energy electron diffraction was used to study growth modes and the epitaxial relationship of the ZnS layers to the substrates. Transmission electron microscopy was used to study the crystal-defect structures. Secondary ion mass spectroscopy was used to determine the chemical profiles of the heteroepitaxial interfaces of ZnS layers grown on arsenic-passivated surfaces. One of the main results demons...

BeTe was epitaxially grown on vicinal Si(1 0 0) substrates for the first time. Reflection high energy electron diffraction (RHEED) was used to study the initial growth mode and the surface structure. Transmission electron microscopy (TEM) was used to study the crystal quality. The material was grown on both bare and arsenic covered vicinal Si(1 0 0) surfaces. Smooth and well-ordered surface structures readily formed during the growth. Low stacking-fault densities were obtained in the BeTe epilayers. The potential applications of the new material are discussed briefly.

The architecture of cellular proteins connected to form signaling pathways in response to internal and external cues is much more complex than a group of simple protein-protein interactions. Post translational modifications on proteins (e.g., phosphorylation of serine, threonine and tyrosine residues on proteins) initiate many downstream signaling events leading to protein-protein interactions and subsequent activation of signaling cascades leading to cell proliferation, cell differentiation and cell death. As evidenced by a rapidly expanding mass spectrometry database demonstrating protein phosphorylation at specific motifs, there is currently a large gap in understanding the functional significance of phosphoproteins with respect to their specific protein connections in the signaling cascades. A comprehensive map that interconnects phospho-motifs in pathways will enable identification of nodal protein interactions that are sensitive signatures indicating a disease phenotype from the physiological hemostasis and provide clues into control of disease. Using a novel phosphopeptide microarray technology, we have mapped endogenous tyrosine-phosphoproteome interaction networks in breast cancer cells mediated by signaling adaptor protein GRB2, which transduces cellular responses downstream of several RTKs through the Ras-ERK signaling cascade. We have identified several previously reported motif specific interactions and novel interactions. The peptide microarray data indicate that various phospho-motifs on a single protein are differentially regulated in various cell types and shows global downregulation of phosphoprotein interactions specifically in cells with metastatic potential. The study has revealed novel phosphoprotein mediated signaling networks, which warrants further detailed analysis of the nodes of protein-protein interaction to uncover their biomarker or therapeutic potential.

Periodic and transient operation of catalytic CO oxidation reactors has been studied more than any other reaction system. Large increases in global rates of reaction are observed with noble metal catalysts using forced cycling of the feed concentration between oxygen and carbon monoxide at frequencies between 0.02 Hz and 1 Hz. Smaller increases in rate of reaction are seen with metal oxide catalysts with much lower resonance frequencies. We review the past research in the area and outline the challenges and the unanswered questions for future research.

The diverse functions of microRNA (miRNA) molecules have drawn broad and intensive interest in various biological fields, biomedical applications, and technology development. Which are endogeneous cellular short RNA molecules found in the cytoplasm as well as in various serum fluids. miRNAs are transcriptional and translational regulatory molecules active in cell division, growth, and apoptosis (1). Dysregulated expression of miRNAs has been implicated in various disease states and has been tested as biomarker candidates (2-4). miRNAs are endogeneous cellular short RNA molecules found in the cytoplasm as well as in various serum fluids. miRNAs are transcriptional and translational regulatory molecules active in cell division, growth, and apoptosis (Bartel, Cell 116:281-97, 2004). Dysregulated expression of miRNAs has been implicated in various disease states and has been tested as biomarker candidates (He et al., Nature 435:828-833, 2005; Lu et al., Nature 435:834-838, 2005; O'Donnell, et al., Nature 435:839-843, 2005). In this chapter, we describe the methods using μParaflo(®) microfluidic oligonucleotide microarray technology for applications in miRNA profiling. One unique feature of this technology is the flexibility that provides users with the freedom to select sequence content either for focused studies wherein only the most relevant sequences are included or for discovery studies wherein the most updated sequence content such as those newly derived from deep sequencing. This chapter provides detailed information from experimental design to sample preparation, as well as data analysis for a miRNA array experiment.

The diverse functions of microRNA (miRNA) molecules have drawn broad and intensive interest in various biological fields, biomedical applications, and technology development. Which are endogeneous cellular short RNA molecules found in the cytoplasm as well as in various serum fluids. miRNAs are transcriptional and translational regulatory molecules active in cell division, growth, and apoptosis (1). Dysregulated expression of miRNAs has been implicated in various disease states and has been tested as biomarker candidates (2-4). miRNAs are endogeneous cellular short RNA molecules found in the cytoplasm as well as in various serum fluids. miRNAs are transcriptional and translational regulatory molecules active in cell division, growth, and apoptosis (Bartel, Cell 116:281-97, 2004). Dysregulated expression of miRNAs has been implicated in various disease states and has been tested as biomarker candidates (He et al., Nature 435:828-833, 2005; Lu et al., Nature 435:834-838, 2005; O'Donnell, et al., Nature 435:839-843, 2005). In this chapter, we describe the methods using μParaflo(®) microfluidic oligonucleotide microarray technology for applications in miRNA profiling. One unique feature of this technology is the flexibility that provides users with the freedom to select sequence content either for focused studies wherein only the most relevant sequences are included or for discovery studies wherein the most updated sequence content such as those newly derived from deep sequencing. This chapter provides detailed information from experimental design to sample preparation, as well as data analysis for a miRNA array experiment.