Springer protocols feed by Genetics/Genomics 
This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.
This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Crystallization of Mouse RIG-I ATPase Domain: In Situ Proteolysis
RIG-I is a key pattern recognition receptor that recognizes cytoplasmic viral RNA. Upon ligand binding, it undergoes a conformational change that induces an active signaling conformation. However, the details of this conformational change remain elusive until high-resolution crystal structures of different functional conformations are available. X-ray crystallography is a powerful tool to study structure–function relationships, but crystallization is often the limiting step of the method. Here, we describe the in situ in-drop proteolysis of RIG-I that yielded crystals of the ATPase domain of mouse RIG-I suitable for ...
Source: Springer protocols feed by Genetics/Genomics - June 24, 2014 Category: Genetics & Stem Cells Source Type: news
Enzymatic Synthesis and Purification of a Defined RIG-I Ligand
Receptor-based nucleic acid sensing constitutes one of the most fundamental mechanisms of our innate immune system to sense viral infection. RIG-I is a cytosolic RNA helicase that senses the presence of 5′ triphosphate RNA species, a common feature of many negative strand RNA viruses. We here describe a protocol to enzymatically synthesize and to purify a defined RIG-I ligand that can be used to study RIG-I activation in vitro and in vivo. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - June 24, 2014 Category: Genetics & Stem Cells Source Type: news
Detection of RNA Modifications by HPLC Analysis and Competitive ELISA
Over 100 different RNA modifications exist that are introduced posttranscriptionally by enzymes at specific nucleotide positions. Ribosomal RNA (rRNA) and transfer RNA (tRNA) exhibit the most and diverse modifications that presumably optimize their structure and function. In contrast, oxidative damage can lead to random modifications in rRNA and messenger RNA (mRNA) that strongly impair functionality. RNA modifications have also been implicated in avoiding self-RNA recognition by the immune system or immune evasion by pathogens. Here, we describe the detection of RNA modifications by HPLC analysis and competitive ELISA. (S...
Source: Springer protocols feed by Genetics/Genomics - June 24, 2014 Category: Genetics & Stem Cells Source Type: news
MicroRNA Methodology: Advances in miRNA Technologies
There is an emerging trend in microRNA research and thus substantial progress in microRNA technologies. In this chapter we provide insights into the main microRNA specific methodologies and critical steps of microRNA expression profiling, target gene identification, and functional confirmation of microRNA effects up to in vivo application of antagomirs. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - June 24, 2014 Category: Genetics & Stem Cells Source Type: news
A Modular Approach to Suppression Assays: TLR Ligands, Conditioned Medium, and Viral Infection
The suppressive function of regulatory T-cells (Treg) requires precise control to allow an efficient adaption of the T-cell response to the requirements of the immune defense. In the setting of infection, an abrogation of the suppressive effect of Treg on the activation and proliferation of T-effector (Teff) cells is a central precondition to allow fast and efficient clearance of the infectious agent. Experimentally, the suppressive function of Treg on Teff can be indirectly measured in coculture proliferation assays. This versatile tool provides a readout of T cell proliferation in the presence of Treg through the measure...
Source: Springer protocols feed by Genetics/Genomics - June 24, 2014 Category: Genetics & Stem Cells Source Type: news
Mapping of Optimal CD8 T Cell Epitopes
Defining the optimal epitope of a CD8 T cell response towards a certain antigen is a multistep procedure that requires the performance of peptide truncation design, ELISPOT peptide titration assays, and assessing the HLA class I restriction of the defined epitope via intracellular cytokine staining assays with B cell lines and epitope-specific CD8 T cell lines. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - June 24, 2014 Category: Genetics & Stem Cells Source Type: news
Repositioning Monocyte TFRN into Fibroblasts
Repositioning of cell-specific Transcription Factor Regulatory Networks (TFRNs) into another cell type is a pragmatic approach to elicit desired functions in impaired cells and also to investigate transcription regulations underlying cellular functions. However, a systematic method to identify a set of transcription factors (TFs) which can efficiently promote TFRN in a foreign cell type is lacking. Here, we demonstrate a perturbation-matrix method for the identification and repositioning of monocyte TFRNs in dermal fibroblasts. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - June 16, 2014 Category: Genetics & Stem Cells Source Type: news
Analysis of TFRNs Associated with Steroid Hormone-Related Cancers
Estrogen and androgen are important endocrine hormones that control physiological functions in reproductive organs and play roles in the generation and/or progression of steroid hormone-related cancers. Their cognate receptors function as hormone-dependent transcription factors, which directly regulate the expression of their target genes. Genome-wide analysis of hormone receptor-related networks will provide new insights into the understanding of the molecular mechanism orchestrated by estrogen and androgen receptors, and will enable the development of new methods for the diagnosis and treatment of steroid hormone-related...
Source: Springer protocols feed by Genetics/Genomics - June 16, 2014 Category: Genetics & Stem Cells Source Type: news
Transcriptional Regulation in Adipogenesis Through PPARγ-Dependent and -Independent Mechanisms by Prostaglandins
Adipogenesis is controlled by complex mechanisms, and transcription factors are involved in its regulation. PPARγ is a ligand-dependent transcription factor and the most important one for adipogenesis. Although prostaglandin (PG) D2 metabolites have been reported as being the ligands of PPARγ, the endogenous PPARγ ligand in adipocytes remains unclear. Here, we show the methods for the general analysis of adipocyte differentiation and the protocols for promoter analysis, fluorescence EMSA, and chromatin immunoprecipitation assay for the transcriptional regulation of the SREBP-1c-activated lipocalin-type PG...
Source: Springer protocols feed by Genetics/Genomics - June 16, 2014 Category: Genetics & Stem Cells Source Type: news
Analysis of NFATc1-Centered Transcription Factor Regulatory Networks in Osteoclast Formation
Osteoclasts are bone-resorbing cells that differentiate from the macrophage/monocyte lineage. The master transcription factor NFATc1 has a central role in the process of this differentiation. Thus, it is important to understand the NFATc1-centered transcription factor regulatory networks (TFRNs) in terms of the mechanisms of NFATc1 expression and activation as well as the gene expression regulated by NFATc1. The Genome Network Project has provided a unique opportunity for the analysis of NFATc1-centered TFRNs in osteoclasts. Here we introduce a report on the application of the methods established by the project for osteocl...
Source: Springer protocols feed by Genetics/Genomics - June 16, 2014 Category: Genetics & Stem Cells Source Type: news
Identification of Transcription Factors Activated in Thymic Epithelial Cells During Embryonic Thymus Development
Differentiation of many immune-related cells is controlled by the expression levels and the activation status of transcription factors (TFs). We here describe a method to identify candidate TFs activated during the development of thymic epithelial cells (TECs) in the embryo. RNAs are isolated from fetal thymic organ cultures of wild-type and mutant mice and are subsequently analyzed by using a combination of comprehensive expression analysis and in silico data analysis in order to predict the TFs that might be activated. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - June 16, 2014 Category: Genetics & Stem Cells Source Type: news
Human Genome Network Platform: A Resource for TFRN Analysis
Genome Network Project (GNP) (Carninci et al., Science 309:1559–1563, 2005) Platform was developed as an integrated database, opening to the public the research findings within the GNP initiatives. Since the first release in 2006, it has gained a large amount of access from all over the world with public favor. The platform is unique and useful in that various types of experimental data for transcriptome analysis are intensively collected, organized, integrated, and visualized with major public datasets; and it can be freely accessed through a single interface with advanced search functionalities. This chapter descri...
Source: Springer protocols feed by Genetics/Genomics - June 16, 2014 Category: Genetics & Stem Cells Source Type: news
Modeling and Simulation Using CellDesigner
In silico modeling and simulation are effective means to understand how the regulatory systems function in life. In this chapter, we explain how to build a model and run the simulation using CellDesigner, adopting the standards such as SBML and SBGN. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - June 16, 2014 Category: Genetics & Stem Cells Source Type: news
Identification of the Minimal Connected Network of Transcription Factors by Transcriptomic and Genomic Data Integration
Thanks to high-throughput experiments, biological conditions can be investigated at both the entire genomic and transcriptomic levels. In addition, protein–protein interaction (PPI) data are widely available for well-studied organisms, such as human. In this chapter, we will present an integrative approach that makes use of these data to find the PPI module involving the key regulated transcription factors shared by a number of given conditions. These conditions could be for instance different cancer types. Briefly, for the studied conditions, we need to identify commonly affected chromosomal regions subjected to cop...
Source: Springer protocols feed by Genetics/Genomics - June 16, 2014 Category: Genetics & Stem Cells Source Type: news
Real-Time PCR Detection of Mycoplasma pneumoniae in the Diagnosis of Community-Acquired Pneumonia
Polymerase chain reaction is a useful technique in microbial diagnostics to detect and quantify DNA or RNA of low abundance. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - April 17, 2014 Category: Genetics & Stem Cells Source Type: news
