Springer protocols feed by Genetics/Genomics 
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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.Surface Functionalization for Immobilization of Probes on Microarrays
The microarray technology has been a tremendous advance in molecular-based testing methods for biochemical and biomedical applications. As a result, the immobilization techniques and grafting chemistries of biochemical molecules have experienced great progress. The particularities of the grafting techniques adapted to the microarray development will be presented here. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - December 1, 2015 Category: Genetics & Stem Cells Source Type: news
Overview of Microarray Technology
Microarray technology, with its high-throughput advantage, has been applied to analyze various biomaterials, such as nucleic acids, proteins, glycans, peptides, and cells. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - December 1, 2015 Category: Genetics & Stem Cells Source Type: news
PAR-CLIP: A Method for Transcriptome-Wide Identification of RNA Binding Protein Interaction Sites
During post-transcriptional gene regulation (PTGR), RNA binding proteins (RBPs) interact with all classes of RNA to control RNA maturation, stability, transport, and translation. Here, we describe Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), a transcriptome-scale method for identifying RBP binding sites on target RNAs with nucleotide-level resolution. This method is readily applicable to any protein directly contacting RNA, including RBPs that are predicted to bind in a sequence- or structure-dependent manner at discrete RNA recognition elements (RREs), and those that are though...
Source: Springer protocols feed by Genetics/Genomics - October 16, 2015 Category: Genetics & Stem Cells Source Type: news
Use of the pBUTR Reporter System for Scalable Analysis of 3′ UTR-Mediated Gene Regulation
Posttranscriptional control of mRNA subcellular localization, stability, and translation is a central aspect of gene regulation and expression. Much of this control is mediated via recognition of a given mRNA transcript’s 3′ untranslated region (UTR) by microRNAs and RNA-binding proteins. Here we describe how a novel, scalable piggyBac-based vector, pBUTR, can be utilized for analysis of 3′ UTR-mediated posttranscriptional gene regulation (PTGR) both in vitro and in vivo. This vector is specifically designed to express a selection marker, a control reporter, and an experimental reporter from three indepen...
Source: Springer protocols feed by Genetics/Genomics - October 16, 2015 Category: Genetics & Stem Cells Source Type: news
Studying Isoform-Specific mRNA Recruitment to Polyribosomes with Frac-seq
Gene expression profiling is widely used as a measure of the protein output of cells. However, it is becoming more evident that there are multiple layers of post-transcriptional gene regulation that greatly impact protein output (Battle et al., Science 347:664–667, 2014; Khan et al., Science 342:1100–1104, 2013; Vogel et al., Mol Syst Biol 6:400, 2010). Alternative splicing (AS) impacts the expression of protein coding genes in several ways. Firstly, AS increases exponentially the coding-capacity of genes generating multiple transcripts from the same genomic sequence. Secondly, alternatively spliced mRNAs are s...
Source: Springer protocols feed by Genetics/Genomics - October 16, 2015 Category: Genetics & Stem Cells Source Type: news
Exploring Ribosome Positioning on Translating Transcripts with Ribosome Profiling
Recent technological advances (e.g., microarrays and massively parallel sequencing) have facilitated genome-wide measurement of many aspects of gene regulation. Ribosome profiling is a high-throughput sequencing method used to measure gene expression at the level of translation. This is accomplished by quantifying both the number of translating ribosomes and their locations on mRNA transcripts [1]. The inventors of this approach have published several methods papers detailing its implementation and addressing the basics of ribosome profiling data analysis [2–4]. Here we describe our lab’s procedure, which diffe...
Source: Springer protocols feed by Genetics/Genomics - October 16, 2015 Category: Genetics & Stem Cells Source Type: news
Studying the Translatome with Polysome Profiling
Polysome fractionation by sucrose density gradient centrifugation followed by analysis of RNA and protein is a technique that allows to understand the changes in translation of individual mRNAs as well as genome-wide effects on the translatome. Here, we describe the polysome profiling technique and RNA as well as protein isolation procedures from sucrose fractions. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - October 16, 2015 Category: Genetics & Stem Cells Source Type: news
Transcriptional Regulation with CRISPR/Cas9 Effectors in Mammalian Cells
We describe the application of a CRISPR-Cas9 effector system from Streptococcus pyogenes for transcriptional regulation in mammalian cells resulting in activation or repression of transcription. We present methods for appropriate target site selection, sgRNA design, and delivery of dCas9 and dCas9-effector system components into cells through lentiviral transgenesis to modulate transcription. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - October 16, 2015 Category: Genetics & Stem Cells Source Type: news
A Computational Approach for the Discovery of Protein–RNA Networks
Protein–RNA interactions play important roles in a wide variety of cellular processes, ranging from transcriptional and posttranscriptional regulation of genes to host defense against pathogens. In this chapter we present the computational approach catRAPID to predict protein–RNA interactions and discuss how it could be used to find trends in ribonucleoprotein networks. We envisage that the combination of computational and experimental approaches will be crucial to unravel the role of coding and noncoding RNAs in protein networks. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - October 16, 2015 Category: Genetics & Stem Cells Source Type: news
Introduction to Bioinformatics Resources for Post-transcriptional Regulation of Gene Expression
Untranslated regions (UTRs) and, to a lesser extent, coding sequences of mRNAs are involved in defining the fate of the mature transcripts through the modulation of three primary control processes, mRNA localization, degradation and translation; the action of trans-factors such as RNA-binding proteins (RBPs) and noncoding RNAs (ncRNAs) combined with the presence of defined sequence and structural cis-elements ultimately determines translation levels. Identifying functional regions in UTRs and uncovering post-transcriptional regulators acting upon these regions is thus of paramount importance to understand the spectrum of r...
Source: Springer protocols feed by Genetics/Genomics - October 16, 2015 Category: Genetics & Stem Cells Source Type: news
Simultaneous Optogenetic Stimulation of Individual Pharyngeal Neurons and Monitoring of Feeding Behavior in Intact C. elegans
Optogenetic approaches have proven powerful for examining the role of neural circuits in generating behaviors, especially in systems where electrophysiological manipulation is not possible. Here we describe a method for optogenetically manipulating single pharyngeal neurons in intact C. elegans while monitoring pharyngeal behavior. This approach provides bidirectional and dynamic control of pharyngeal neural activity simultaneously with a behavioral readout and has allowed us to test hypotheses about the roles of individual pharyngeal neurons in regulating feeding behavior. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - October 2, 2015 Category: Genetics & Stem Cells Source Type: news
Microbial Rhodopsin Optogenetic Tools: Application for Analyses of Synaptic Transmission and of Neuronal Network Activity in Behavior
Optogenetics was introduced as a new technology in the neurosciences about a decade ago (Zemelman et al., Neuron 33:15–22, 2002; Boyden et al., Nat Neurosci 8:1263–1268, 2005; Nagel et al., Curr Biol 15:2279–2284, 2005; Zemelman et al., Proc Natl Acad Sci USA 100:1352–1357, 2003). It combines optics, genetics, and bioengineering to render neurons sensitive to light, in order to achieve a precise, exogenous, and noninvasive control of membrane potential, intracellular signaling, network activity, or behavior (Rein and Deussing, Mol Genet Genomics 287:95–109, 2012; Yizhar et al., Neuron 71:9&nda...
Source: Springer protocols feed by Genetics/Genomics - October 2, 2015 Category: Genetics & Stem Cells Source Type: news
Observing and Quantifying Fluorescent Reporters
Genetically encoded fluorescent reporters take advantage of C. elegans’ transparency to allow noninvasive, in vivo observation and recording of physiological processes in intact animals. Here, I discuss the basic microscope components required to observe, image, and measure fluorescent proteins in live animals for students and researchers who work with C. elegans but have limited experience with fluorescence imaging and analysis. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - October 2, 2015 Category: Genetics & Stem Cells Source Type: news
Creating Genome Modifications in C. elegans Using the CRISPR/Cas9 System
The clustered, regularly interspaced, short, palindromic repeat (CRISPR)-associated (CAS) nuclease Cas9 has been used in many organisms to generate specific mutations and transgene insertions. Here we describe a method using the S. pyogenes Cas9 in C. elegans that provides a convenient and effective approach for making heritable changes to the worm genome. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - October 2, 2015 Category: Genetics & Stem Cells Source Type: news
Transposon-Assisted Genetic Engineering with Mos1-Mediated Single-Copy Insertion (MosSCI)
Transgenesis in model organisms is necessary to determine the function, expression, and subcellular localization of gene products. In Caenorhabditis elegans, injected DNA can be propagated as multicopy extrachromosomal arrays but transgenes in arrays are mosaic, over-expressed in some tissues and silenced in the germline. Here, a method to insert a transgene into a specific genomic location called Mos1-mediated single-copy insertion (MosSCI) is described. Single-copy insertion allows transgene expression at levels that approximate endogenous gene expression as well as expression in the germline. (Source: Springer protocols...
Source: Springer protocols feed by Genetics/Genomics - October 2, 2015 Category: Genetics & Stem Cells Source Type: news
