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检索条件"主题词=Programming Language"

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Methods for RNA Profiling of Gravi-Responding Plant Tissues

Methods for RNA Profiling of Gravi-Responding Plant Tissues

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作者： Jyoti Dalal Eric Land Naresh Vasani Luyan He Caroline Smith Maria Rodriguez-Welsh Imara Y. Perera Heike Sederoff

Plant transcriptional responses to gravity stimulation by reorientation are among the fastest measured in any tissue or species. Upon reorientation, changes in abundance of specific mRNAs can be measured within seconds or minutes, for plastid or nuclear encoded genes, respectively. Identifying fast gravity-induced transcripts has been made possible by the development of high-throughput technology for qualitative and quantitative RNA analysis. RNA profiling has undergone further rapid development due to its enormous potential in basic sciences and medical applications. We describe here the current and most widely used methods to profile the changes in an entire transcriptome by high-throughput sequencing of RNA fractions (RNAseq) and single gene transcript analysis using real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). less

关键词： PCR RNA-seq Total RNA Extraction Next Generation Sequencing Reverse Transcription PCR Real-time PCR Transcription Profiling RNA Extraction Library Preparation High-Throughput Sequencing Homogenization Quantitative Reverse Transcription PCR RNA Quality Analysis RNA Electrophoresis Fractionation Size-exclusion Chromatography Nucleic Acid Microarray Agarose Gel Electrophoresis Trinity Sterilization Data analysis DNA Ligation programming language Sequencing RNA miRNA Transcriptome Transcript sRNA

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Enrichment Analysis of miRNA Targets

Enrichment Analysis of miRNA Targets

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作者： Jianzhen Xu Chi-Wai Wong

MicroRNAs (miRNAs) are a class of ∼22-nucleotide endogenous noncoding RNAs which regulate target gene expression via repressing translation or promoting mRNAs degradation. Any individual mammalian miRNA often has more than a hundred predicted mRNA targets and that close to one thirds of all mRNA transcripts bear one or more conserved miRNA binding sites in their 3′-untranslated region. Enrichment analysis of miRNA targets has become a standard technique to elucidating hierarchical functions of miRNAs in gene regulatory networks. In this protocol, we discuss analytical methods and use of computational tools in a step-by-step manner. Important details are also provided to help researchers choose more appropriate tools for a given type of analysis. Available Web-based resources for enrichment analysis of miRNA targets are summarized. less

关键词： Microarray Gene Expression Profiling Reverse Transcription PCR Northern Blot Proteomics Recombinant Protein Expression programming language Enrichment analysis Functional annotation MicroRNA Web resources Computational prediction MicroRNA targets

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Statistical Analysis of ATM-Dependent Signaling in Quantitative Mass Spectrometry Phosphoproteomics

Statistical Analysis of ATM-Dependent Signaling in Quantitat...

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作者： Ashley J. Waardenberg

Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase, which when perturbed is associated with modified protein signaling that ultimately leads to a range of neurological and DNA repair defects. Recent advances in phospho-proteomics coupled with high-resolution mass-spectrometry provide new opportunities to dissect signaling pathways that ATM utilize under a number of conditions. This chapter begins by providing a brief overview of ATM function, its various regulatory roles and then leads into a workflow focused on the use of the statistical programming language R, together with code, for the identification of ATM-dependent substrates in the cytoplasm. This chapter cannot cover statistical properties in depth nor the range of possible methods in great detail, but instead aims to equip researchers with a set of tools to perform analysis between two conditions through examples with R functions. less

关键词： Statistical Calculation Mass Spectrometry UniProtKB programming language Bioinformatics Phosphorylation Phosphoproteomics Ranked product analysis

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The Comprehensive and Reliable Detection of Secondary Metabolites in

The Comprehensive and Reliable Detection of Secondary Metabo...

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作者： Bernhard Seidl Christoph Bueschl Rainer Schuhmacher

A method based on reversed phase high-performance liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry (RP-HPLC-ESI-HRMS) for the comprehensive and reliable detection of secondary metabolites of Trichoderma reesei cultured in synthetic minimal liquid medium is presented. A stable isotope-assisted (SIA) workflow is used, which allows the automated, comprehensive extraction of truly fungal metabolite-derived LC-MS signals from the acquired chromatographic data. The subsequent statistical data analysis and a typical outcome of such a metabolomics data evaluation are shown by way of example in a previously published study on the influence of the pleiotropic regulator transcription factor Xylanase promoter binding protein 1 (Xpp1) in T. reesei on secondary metabolism. less

关键词： Metabolomics Statistical Calculation Isotope Labeling Mass Spectrometry Reversed-phase HPLC Electrospray Ionization (ESI) High Resolution Mass Spectrometry Liquid Chromatography-mass Spectrometry Freeze-thaw Method Principal Component Analysis Database Comparison Database Search HPLC Data analysis programming language Trichoderma Stable isotope-assisted workflow

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Engineering of human cardiac muscle electromechanically matured to an adult-like phenotype

Engineering of human cardiac muscle electromechanically matu...

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作者： Kacey Ronaldson-Bouchard Keith Yeager Diogo Teles Timothy Chen Stephen Ma LouJin Song Kumi Morikawa Holly M. Wobma Alessandro Vasciaveo Edward C. Ruiz Masayuki Yazawa Gordana Vunjak-Novakovic

The application of tissue-engineering approaches to human induced pluripotent stem (hiPS) cells enables the development of physiologically relevant human tissue models for in vitro studies of development, regeneration, and disease. However, the immature phenotype of hiPS-derived cardiomyocytes (hiPS-CMs) limits their utility. We have developed a protocol to generate engineered cardiac tissues from hiPS cells and electromechanically mature them toward an adult-like phenotype. This protocol also provides optimized methods for analyzing these tissues’ functionality, ultrastructure, and cellular properties. The approach relies on biological adaptation of cultured tissues subjected to biomimetic cues, applied at an increasing intensity, to drive accelerated maturation. hiPS cells are differentiated into cardiomyocytes and used immediately after the first contractions are observed, when they still have developmental plasticity. This starting cell population is combined with human dermal fibroblasts, encapsulated in a fibrin hydrogel and allowed to compact under passive tension in a custom-designed bioreactor. After 7 d of tissue formation, the engineered tissues are matured for an additional 21 d by increasingly intense electromechanical stimulation. Tissue properties can be evaluated by measuring contractile function, responsiveness to electrical stimuli, ultrastructure properties (sarcomere length, mitochondrial density, networks of transverse tubules), force–frequency and force–length relationships, calcium handling, and responses to β-adrenergic agonists. Cell properties can be evaluated by monitoring gene/protein expression, oxidative metabolism, and electrophysiology. The protocol takes 4 weeks and requires experience in advanced cell culture and machining methods for bioreactor fabrication. We anticipate that this protocol will improve modeling of cardiac diseases and testing of drugs. less

关键词： Biomedical Engineering Cell And Tissue Culture Electrophysiology Tissue Engineering Immunostaining Flow Cytometry TEM Gene Expression Analysis Principal Component Analysis Long-term Culture Cross-linking MATLAB Confocal Microscopy High-throughput Screening Injection Molding Degassing ImageJ Calcium Imaging Paraffin Embedding Single-cell Sequencing MetaMorph Microscopy Image Analysis programming language Homo sapiens Mycoplasma Bos taurus Hippocampus Triticum aestivum Stem-cell differentiation Cardiovascular models Anti-sarcomeric α-actinin Anti-cTnT Anti-OxPhos Anti-mouse Anti-rabbit Anti-vimentin (VIM) Anti-human

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Statistical Evaluation of Genetic Footprinting Data

Statistical Evaluation of Genetic Footprinting Data

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作者： Gábor Balázsi

As transposomics is extended to genome scale, appropriate statistical methods need to be developed to assign significance to gene essentiality. In this chapter, the author presents a set of steps that, together with genome-scale insertion data and the complete genome sequence of a prokaryote, can be used to classify the genes of the organism as either “essential” or “nonessential.” less

关键词： Mutagenesis programming language genetics significance essentiality insertion Poisson distribution transposomics

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WormBase

WormBase

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作者： Todd W. Harris Lincoln D. Stein

WormBase is a comprehensive repository for information on Caenorhabditis elegans and related nematodes. Although the primary web-based interface of WormBase (http://***/ ) is familiar to most C. elegans researchers, WormBase also offers powerful data-mining features for addressing questions of comparative genomics, genome structure, and evolution. In this chapter, we focus on data mining at WormBase through the use of flexible web interfaces, custom queries, and scripts. The intended audience includes users wishing to query the database beyond the confines of the web interface or fetch data en masse. No knowledge of programming is necessary or assumed, although users with intermediate skills in the Perl scripting language will be able to utilize additional data-mining approaches. less

关键词： Data Mining Genomics Genome Visualization RNA Interference Microarray programming language Caenorhabditis elegans bioinformatics WormBase AceDB AcePerl

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Synthetic Networks: Oscillators and Toggle Switches for

Synthetic Networks: Oscillators and Toggle Switches for

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作者： Nicolas Perry Alexander J. Ninfa

Bacterial synthetic gene networks are constructed by manipulating the regulation of genes inside a cell, with the purpose of eliciting novel regulatory behaviors. The methods for manipulating genes and gene regulation in E. coli are well established, making it the preferred host for basic studies of synthetic networks. We focus our work on constructing two kinds of synthetic gene networks: toggle switches (bistable systems) and oscillators. Toggle switches are capable of exhibiting two stable steady states of gene expression (OFF and ON) without stable intermediate states; the steady state reached by the system depends on the previous history of the system. Biological oscillators exhibit regular cycles in gene expression around an unstable steady state. Studying these two kinds of synthetic networks helps advance our understanding of natural bistable systems and oscillators, such as the circadian oscillators controlling gene expression in many types of cells, and the genetic systems controlling the cell cycle and differentiation in metazoans. less

关键词： Restriction Enzyme Digestion Electroporation Colony PCR Microbial Culture PCR Gel Electrophoresis Transillumination Bacterial Transformation DNA Ligation programming language Escherichia coli Recombinant DNA Genetic circuit Genetic oscillator Genetic switch Genetic bistability Continuous culture automation

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Lectin Microarrays for Glycoprotein Analysis

Lectin Microarrays for Glycoprotein Analysis

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作者： Kanoelani T. Pilobello Lara K. Mahal

Glycosylation is one of the most common posttranslational modifications, with more than half of all known proteins thought to be glycoproteins. Alterations in glycosylation play a role in a diverse set of biological phenomena including tumor cell metastasis, intracellular communication, and inflammation. The complexity of glycosylation at the molecular level and the lack of rapid analytical tools complicate the study of glycan function. We have recently developed a lectin microarray for the high-throughput analysis of glycosylation. Lectins are carbohydrate-binding proteins that have been used for decades as a detection method for glycans. By placing the lectins in a microarray format and using standard microarray printing and scanning technology, we have created a simple yet powerful technique for glycan profiling. less

关键词： Microarray Dialysis Data analysis Python programming language Bos taurus Lotus tetragonolobus Lens culinaris Glycine max Solanum tuberosum Triticum array glycomics carbohydrate analysis glycan profiling

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Analysis of the Mobility of DNA Double-Strand Break-Containing Chromosome Domains in Living Mammalian Cells

Analysis of the Mobility of DNA Double-Strand Break-Containi...

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作者： Przemek M. Krawczyk Jan Stap Ron A. Hoebe Carel H. Oven Roland Kanaar Jacob A. Aten

DNA double-strand breaks (DSBs) are among the most dangerous types of DNA damage. Unrepaired, DSBs may lead to cell death, and when misrejoined, they can result in potentially carcinogenic chromosome rearrangements. The induction of DSBs and their repair take place in a chromatin microenvironment. Therefore, understanding and describing the dynamics of DSB-containing chromatin is of crucial importance for understanding interactions among DSBs and their repair. Recent developments have made it possible to study ionizing radiationinduced foci of DSB repair proteins in vivo. In this chapter, we describe techniques that can be applied to visualize and analyze the spatio-temporal dynamics of DSB-containing chromatin domains in mammalian cell nuclei. Analogous procedures may also be applied to the analysis of mobility of other intranuclear structures in living cells. less

关键词： Transfection Time Lapse Microscopy Image Processing Cell And Tissue Culture Western Blot Microscopy Fluorescence Microscopy programming language DNA double-strand breaks Live cells Chromosome domain mobility

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