3,984 results match your criteria Methods [Journal]


Real-time monitoring of protein-induced DNA conformational changes using single-molecule FRET.

Methods 2019 Feb 15. Epub 2019 Feb 15.

B CUBE - Center for Molecular Bioengineering, TU Dresden, Tatzberg 41, 01307 Dresden, Germany. Electronic address:

Apart from being storage devices for genetic information, nucleic acids can provide regulatory structures through evolutionarily optimized sequences. The interaction of proteins binding specifically to such sequences and resulting secondary structures, or the exposure of single-stranded DNA add a versatile regulatory framework for cells. Biochemical and structural biology experiments have revealed important underlying concepts of protein-DNA interactions but are often limited by ensemble averaging or static information. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.02.011DOI Listing
February 2019

Deep-Resp-Forest: A deep forest model to predict anti-cancer drug response.

Methods 2019 Feb 14. Epub 2019 Feb 14.

Institute of Fundamental and Frontier Sciences, University of Electronic Science and Technology of China, Chengdu, China. Electronic address:

The identification of therapeutic biomarkers predictive of drug response is crucial in personalized medicine. A number of computational models to predict response of anti-cancer drugs have been developed as the establishment of several pharmacogenomics screening databases. In our study, we proposed a deep cascaded forest model, Deep-Resp-Forest, to classify the anti-cancer drug response as "sensitive" or "resistant". Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.02.009DOI Listing
February 2019

Use and reliability of multiplex bead-based assays (Luminex) at Containment Level 4.

Methods 2019 Feb 13. Epub 2019 Feb 13.

Public Health England, Porton Down, Salisbury, Wiltshire, SP4 0JG. UK.

In the UK, research on hazard group 4 (HG4) pathogens requires specialised Containment Level 4 (CL4) facilities. These differ from Biosafety Level 4 (BSL4) conditions in that work is conducted in class III microbiological safety cabinets for primary containment instead of using positive pressure suits. This presents unique challenges associated with the physical restrictions of working in a limited space, and prohibits the use of many techniques and specialist equipment. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.02.008DOI Listing
February 2019

Computer-aided detection of mass in digital breast tomosynthesis using a faster region-based convolutional neural network.

Methods 2019 Feb 13. Epub 2019 Feb 13.

Institute of Biomedical Engineering and Instrumentation, Hangzhou Dianzi University, High Education Zone, Hangzhou 310018, China. Electronic address:

Digital breast tomosynthesis (DBT) is a newly developed three-dimensional tomographic imaging modality in the field of breast cancer screening designed to alleviate the limitations of conventional digital mammography-based breast screening methods. A computer-aided detection (CAD) system was designed for masses in DBT using a faster region-based convolutional neural network (faster-RCNN). To this end, a data set was collected, including 89 patients with 105 masses. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.02.010DOI Listing
February 2019
1 Read

Analysis of RNA polymerase II ubiquitylation and proteasomal degradation.

Methods 2019 Feb 12. Epub 2019 Feb 12.

Mechanisms of Transcription Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. Electronic address:

Transcribing RNA polymerase II (RNAPII) is decorated by a plethora of post-translational modifications that mark different stages of transcription. One important modification is RNAPII ubiquitylation, which occurs in response to numerous different stimuli that cause RNAPII stalling, such as DNA damaging agents, RNAPII inhibitors, or depletion of the nucleotide pool. Stalled RNAPII triggers a so-called "last resort pathway", which involves RNAPII poly-ubiquitylation and proteasome-mediated degradation. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.02.005DOI Listing
February 2019
3.645 Impact Factor

Use of the Nuclear Walk-on Methodology to Determine Sites of RNA Polymerase II Initiation and Pausing and Quantify Nascent RNAs in Cells.

Methods 2019 Feb 8. Epub 2019 Feb 8.

Department of Biochemistry, University of Iowa, Iowa City, IA, 52242, United States. Electronic address:

Transcription by RNA polymerase II (Pol II) is controlled during initiation, elongation, and termination by a large variety of transcription factors, the state of chromatin modifications, and environmental conditions. Herein we describe experimental approaches for the examination of Pol II transcription at semi-global and genome-wide scales through analysis of nascent Pol II transcripts. We begin with a description of the nuclear walk-on (NWO) assay, which involves rapid isolation of nuclei in the presence of EDTA, followed by extension of about a quarter of the nascent transcripts with P-CTP. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.02.003DOI Listing
February 2019

Tracking of single tRNAs for translation kinetics measurements in chloramphenicol treated bacteria.

Methods 2019 Feb 8. Epub 2019 Feb 8.

Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden. Electronic address:

Chloramphenicol is a broad-spectrum antibiotic targeting the protein synthesis machinery by binding to the bacterial ribosome. Chloramphenicol has been considered a classic general inhibitor of translation, blocking the accommodation of aa-tRNA into the A site of the large ribosomal subunit. However, recent studies suggest that this proposed mechanism is a simplification and that the effect of chloramphenicol on mRNA translation is much more dynamic. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.02.004DOI Listing
February 2019

Genome-wide analysis of RNA and protein localization and local translation in mESC-derived neurons.

Methods 2019 Feb 8. Epub 2019 Feb 8.

Non-coding RNAs and Mechanisms of Cytoplasmic Gene Regulation, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin, Germany. Electronic address:

The subcellular localization and translation of mRNAs are fundamental biological processes. In neurons, they underlie cell growth and synaptic plasticity, which serves as a foundation of learning and memory. Multiple approaches have been developed to separate neurons on subcellular compartments - cell bodies (soma) and cell extensions (axons and dendrites) - for further biochemical analyses. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.02.002DOI Listing
February 2019

Method to Quantify Cytokines and Chemokines in Mouse Brain Tissue Using Bio-Plex Multiplex Immunoassays.

Methods 2019 Feb 8. Epub 2019 Feb 8.

Bio-Rad Laboratories, Hercules, California, 94547, USA.

This protocol describes how to prepare mouse brain tissue for quantification of multiple inflammatory mediators using a multiplex bead-based immunoassay. It is important to have methods that allow quantification of multiple analytes from small amounts of tissue. Bio-Plex is a Luminex xMAP-based multiplex bead-based immunoassay technology that permits simultaneous analysis of up to 100 analytes from a single tissue sample. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S10462023183023
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http://dx.doi.org/10.1016/j.ymeth.2019.02.007DOI Listing
February 2019
1 Read

Multiplexing Protein and Gene Level Measurements on a Single Luminex Platform.

Methods 2019 Feb 8. Epub 2019 Feb 8.

Thermo Fisher Scientific, Carlsbad, CA 92008, United States. Electronic address:

Background: The ability to simultaneously measure multiple secreted proteins and the corresponding gene expression levels from a single sample is valuable for comprehensive analysis. Bottlenecks to traditional immunoassays and gene expression assays include large sample consumption, time consuming experimental procedures, and complex data analysis.

Method And Results: Here, we demonstrate two high-throughput assays measuring both messenger RNA (mRNA) expression and proteins in a single sample run on a Luminex platform. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.01.018DOI Listing
February 2019

Deriving a sub-nanomolar affinity peptide from TAP to enable smFRET analysis of RNA polymerase II complexes.

Methods 2019 Feb 10. Epub 2019 Feb 10.

Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan. Electronic address:

Our capability to visualize protein complexes such as RNA polymerase II (pol II) by single-molecule imaging techniques has largely been hampered by the absence of a simple bio-orthogonal approach for selective labeling with a fluorescent probe. Here, we modify the existing calmodulin-binding peptide (CBP) in the widely used Tandem Affinity Purification (TAP) tag to endow it with a high affinity for calmodulin (CaM) and use dye-CaM to conduct site-specific labeling of pol II. To demonstrate the single molecule applicability of this approach, we labeled the C-terminus of the Rpb9 subunit of pol II with donor-CaM and a site in TFIIF with an acceptor to generate a FRET (fluorescence resonance energy transfer) pair in the pol II-TFIIF complex. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.02.006DOI Listing
February 2019

Applications of high-throughput sequencing to analyze and engineer ribozymes.

Methods 2019 Feb 6. Epub 2019 Feb 6.

Nucleic Acid Chemistry and Engineering Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa 904 0495, Japan. Electronic address:

A large number of catalytic RNAs, or ribozymes, have been identified in the genomes of various organisms and viruses. Ribozymes are involved in biological processes such as regulation of gene expression and viral replication, but biological roles of many ribozymes still remain unknown. Ribozymes have also inspired researchers to engineer synthetic ribozymes that function as sensors or gene switches. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.02.001DOI Listing
February 2019
1 Read

Measuring RNA polymerase activity genome-wide with high-resolution run-on-based methods.

Methods 2019 Feb 2. Epub 2019 Feb 2.

ERI Biotecmed, Facultad de Biológicas, Universitat de València, C/Dr. Moliner 50, E46100 Burjassot, Spain.

The biogenesis of RNAs is a multi-layered and highly regulated process that involves a diverse set of players acting in an orchestrated manner throughout the transcription cycle. Transcription initiation, elongation and termination factors act on RNA polymerases to modulate their movement along the DNA template in a very precise manner, more complex than previously anticipated. Genome-scale run-on-based methodologies have been developed to study in detail the position of transcriptionally-engaged RNA polymerases. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.01.017DOI Listing
February 2019

Time-resolved analysis of transcription through chromatin.

Methods 2019 Jan 29. Epub 2019 Jan 29.

Fox Chase Cancer Center, Philadelphia, PA 19111, USA; Biology Faculty, Lomonosov Moscow State University, Moscow 119992, Russia. Electronic address:

During transcription along nucleosomal DNA, RNA polymerase II (Pol II) pauses at multiple positions and induces formation of multiple intermediates that aid in maintaining proper chromatin structure. To describe the kinetics of this multiple-step reaction, we utilized a computational model-based approach and KinTek Explorer software to analyze the time courses. Here we describe the stepwise protocol for analysis of the kinetics of transcription through a nucleosome that provides the rate constants for each step of this complex process. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.01.016DOI Listing
January 2019

The development of a multiplex serological assay for avian influenza based on Luminex technology.

Methods 2019 Jan 30. Epub 2019 Jan 30.

Wageningen Bioveterinary Research, Houtribweg 39, 8221 RA Lelystad, the Netherlands. Electronic address:

Avian influenza (AI) is an infectious disease in birds with enormous impact on the poultry sector. AI viruses are divided into different subtypes based on the antigenicity of their surface proteins haemagglutinin (HA) and neuraminidases (NA). In birds, 16 HA subtypes and 9 NA subtypes are detected in different combinations. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.01.012DOI Listing
January 2019
1 Read

A universal method for the functionalization of dyed magnetic microspheres with peptides.

Methods 2019 Jan 30. Epub 2019 Jan 30.

Sensors and Electron Devices Directorate, U.S. Army Research Laboratory, 2800 Powder Mill Rd, Adelphi, MD, United States.

The need for the functionalization of magnetic, water-soluble dyed microspheres with peptides is apparent with the ever-growing biointeraction capabilities and the increased use of dyed microspheres in multiplex, microsphere-based detection assays. This method describes the attachment of any peptide to dyed magnetic microspheres regardless of peptide length, size, or sequence. The method exploits 'click' chemistry with short reaction times in a mixed organic/water system for simultaneous selective surface functionalization and reduction of microsphere dye leaching. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.01.014DOI Listing
January 2019

Validation of monoplex assays detecting antibodies against Corynebacterium diphtheriae and Clostridium tetani toxins, rubella virus and parvovirus B19 for incorporation into Multiplex Serology.

Methods 2019 Jan 28. Epub 2019 Jan 28.

Infections and Cancer Epidemiology, German Cancer Research Center, Heidelberg, Germany. Electronic address:

Serological assays detecting antibodies in serum or plasma samples are useful and versatile instruments to investigate an individual's infection and vaccination history, e.g. for clinical diagnosis, personal risk evaluation, and seroepidemiological studies. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.01.013DOI Listing
January 2019
1 Read

Development and Validation of Magnetic Bead Pentaplex Immunoassay for Simultaneous Quantification of murine serum IgG antibodies to Acellular Pertussis, Diphtheria and Tetanus Antigens used in combination vaccines.

Methods 2019 Jan 25. Epub 2019 Jan 25.

Serum Institute of India Pvt Ltd, 212/2, Hadapsar, Pune, Maharashtra, 411028, India. Electronic address:

We describe here a magnetic bead-based multiplex (pentaplex) immunoassay (MIA) platform developed as an alternative to enzyme-linked immunosorbent assays (ELISA) used in immunogenicity testing of DTaP/TdaP vaccine in animals. MIA simultaneously measures the concentration of serum (IgG) antibodies against B. Pertussis antigens; pertussis toxin, , filamentous hemagglutinin (FHA), pertactin (PRN) and tetanus (T) and diphtheria (D) toxoid in the Tdap vaccine immunized animals. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.01.015DOI Listing
January 2019
2 Reads

Genetic analysis of the RNA polymerase II CTD in Drosophila.

Methods 2019 Jan 24. Epub 2019 Jan 24.

Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA. Electronic address:

The Carboxy-terminal Domain (CTD) of RNA polymerase II (Pol II) plays essential roles in regulating gene expression in eukaryotes. Here, we describe multiple genetic approaches for studying the CTD in Drosophila that complement pre-existing molecular analyses of the Pol II CTD in other experimental models. These approaches will allow one to assess the effects of any CTD mutations in a developmentally complex organism. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.01.010DOI Listing
January 2019
1 Read

Biochemical methods to characterize RNA polymerase II elongation complexes.

Methods 2019 Jan 24. Epub 2019 Jan 24.

Department of Biochemistry and Molecular Biology, Center for Eukaryotic Gene Regulation, Penn State University, University Park, PA 16802, United States. Electronic address:

Transcription of DNA into RNA is critical for all life, and RNA polymerases are enzymes tasked with this activity. In eukaryotes, RNA Polymerase II (RNAPII) is responsible for transcription of all protein coding genes and many non-coding RNAs. RNAPII carries out the remarkable feat of unwinding the stable double-stranded DNA template, synthesizing the transcript and re-forming the double helix behind it with great precision and speed. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.01.011DOI Listing
January 2019
1 Read

"Second-generation" fluorogenic RNA-based sensors.

Methods 2019 Jan 17. Epub 2019 Jan 17.

Department of Chemistry, University of Massachusetts, Amherst, MA 01003, USA. Electronic address:

A fluorogenic aptamer can specifically interact with a fluorophore to activate its fluorescence. These nucleic acid-based fluorogenic modules have been dramatically developed over the past decade, and have been used as versatile reporters in the sensor development and for intracellular imaging. In this review, we summarize the design principles, applications, and challenges of the first-generation fluorogenic RNA-based sensors. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.01.008DOI Listing
January 2019
1 Read

Single-molecule FRET method to investigate the dynamics of transcription elongation through the nucleosome by RNA polymerase II.

Methods 2019 Jan 17. Epub 2019 Jan 17.

Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, United States. Electronic address:

Transcription elongation through the nucleosome is a precisely coordinated activity to ensure timely production of RNA and accurate regulation of co-transcriptional histone modifications. Nucleosomes actively participate in transcription regulation at various levels and impose physical barriers to RNA polymerase II (RNAPII) during transcription elongation. Despite its high significance, the detailed dynamics of how RNAPII translocates along nucleosomal DNA during transcription elongation and how the nucleosome structure dynamically conforms to the changes necessary for RNAPII progression remain poorly understood. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S10462023183029
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http://dx.doi.org/10.1016/j.ymeth.2019.01.009DOI Listing
January 2019
5 Reads

Multiplexed detection and identification of respiratory pathogens using the NxTAG® respiratory pathogen panel.

Methods 2019 Jan 17. Epub 2019 Jan 17.

Pathology and Laboratory Medicine, Northwell Health Laboratories, Lake Success, NY, USA.

The Luminex® NxTAG® Respiratory Pathogen Panel (NxTAG RPP) is an IVD-cleared assay for the simultaneous detection and identification of nucleic acids from 18 respiratory viruses and 2 (or 3 outside of the U.S.) atypical bacterial pathogens in nasopharyngeal swabs. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.01.005DOI Listing
January 2019
2 Reads

Multiplexing techniques for measurement of the immunomodulatory effects of particulate materials: Precautions when testing micro- and nano-particles.

Methods 2019 Jan 17. Epub 2019 Jan 17.

Bio-Rad Laboratories, Hercules, CA 94547, USA.

Particulate materials at nano- and micro-scales have widespread pharmaceutical and medical applications. Understanding the interactions of these materials with biological systems is crucial for the design of clinically-viable biomaterials of high safety profiles. Immunomodulatory effects of particulate materials can be studied via multiplexing techniques that are capable of measuring up to 500 biomarkers in a few microliters of biological samples. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.01.006DOI Listing
January 2019

The genesis and evolution of bead-based multiplexing.

Methods 2019 Jan 17. Epub 2019 Jan 17.

Global Scientific Affairs, Luminex Corporation, 12212 Technology Blvd, Austin, TX, 78727, United States. Electronic address:

Multiplexed analysis has the advantage of allowing for simultaneous detection of multiple analytes in a single reaction vessel which reduces time, labor, and cost as compared to single-reaction-based detection methods. Microsphere-based suspension array technologies, such as the Luminex® xMAP® system, offer high-throughput detection of both protein and nucleic acid targets in multiple assay chemistries. After Luminex's founding in 1995, it quickly became the leader in bead-based multiplexing solutions. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.01.007DOI Listing
January 2019
1 Read

Neomycin-dependent hammerhead ribozymes for the direct control of gene expression in Saccharomyces cerevisiae.

Methods 2019 Jan 9. Epub 2019 Jan 9.

Department of Chemistry, University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany; Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany. Electronic address:

Hammerhead ribozyme-based RNA switches have been proven to be powerful tools for conditional gene regulation in various organisms. We present neomycin-dependent hammerhead ribozymes (HHR) that influence gene expression in a ligand- and dose-dependent manner in S. cerevisiae. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S10462023183027
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http://dx.doi.org/10.1016/j.ymeth.2019.01.004DOI Listing
January 2019
4 Reads

A humanized yeast system to analyze cleavage of prelamin A by ZMPSTE24.

Methods 2019 Jan 6. Epub 2019 Jan 6.

Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD, United States. Electronic address:

The nuclear lamins A, B, and C are intermediate filament proteins that form a nuclear scaffold adjacent to the inner nuclear membrane in higher eukaryotes, providing structural support for the nucleus. In the past two decades it has become evident that the final step in the biogenesis of the mature lamin A from its precursor prelamin A by the zinc metalloprotease ZMPSTE24 plays a critical role in human health. Defects in prelamin A processing by ZMPSTE24 result in premature aging disorders including Hutchinson Gilford Progeria Syndrome (HGPS) and related progeroid diseases. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.01.001DOI Listing
January 2019

Development of Poly(A)-ClickSeq as a tool enabling simultaneous genome-wide poly(A)-site identification and differential expression analysis.

Methods 2019 Jan 6. Epub 2019 Jan 6.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA; Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX, USA. Electronic address:

The use of RNA-seq as a generalized tool to measure the differential expression of genes has essentially replaced the use of the microarray. Despite the acknowledged technical advantages to this approach, RNA-seq library preparation remains mostly conducted by core facilities rather than in the laboratory due to the infrastructure, expertise and time required per sample. We have recently described two 'click-chemistry' based library construction methods termed ClickSeq and Poly(A)-ClickSeq (PAC-seq) as alternatives to conventional RNA-seq that are both cost effective and rely on straightforward reagents readily available to most labs. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.01.002DOI Listing
January 2019
5 Reads

Inhibiting transcription in cultured metazoan cells with actinomycin D to monitor mRNA turnover.

Methods 2019 Jan 6. Epub 2019 Jan 6.

The Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, United States; The Departments of Medicine and Biochemistry, Duke University School of Medicine, Durham, NC 27710, United States. Electronic address:

Decay of transcribed mRNA is a key determinant of steady state mRNA levels in cells. Global analysis of mRNA decay in cultured cells has revealed amazing heterogeneity in rates of decay under normal growth conditions, with calculated half-lives ranging from several minutes to many days. The factors that are responsible for this wide range of decay rates are largely unknown, although our knowledge of trans-acting RNA binding proteins and non-coding RNAs that can control decay rates is increasing. Read More

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http://dx.doi.org/10.1016/j.ymeth.2019.01.003DOI Listing
January 2019

Introduction to "Convergence of Science and Technology: Fluorescent Resolution of Single RNA Molecules".

Authors:
Nils G Walter

Methods 2019 Jan;153:1-2

Francis S. Collins Collegiate Professor of Chemistry, Biophysics & Biological Chemistry, Department of Chemistry, University of Michigan, 930 N. University Ave., Ann Arbor, MI 48109-1055, United States. Electronic address:

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http://dx.doi.org/10.1016/j.ymeth.2018.12.003DOI Listing
January 2019

A fluorescent assay for the genetic dissection of the RNA polymerase II termination machinery.

Authors:
Daniel Reines

Methods 2019 Jan 5. Epub 2019 Jan 5.

Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, United States. Electronic address:

RNA polymerase II is a highly processive enzyme that synthesizes mRNAs and some non-protein coding RNAs. Termination of transcription, which entails release of the transcript and disengagement of the polymerase, requires an active process. In yeast, there are at least two multi-protein complexes needed for termination of transcription, depending upon which class of RNAs are being acted upon. Read More

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http://dx.doi.org/10.1016/j.ymeth.2018.12.011DOI Listing
January 2019

Using time-lapse fluorescence microscopy to study gene regulation.

Authors:
Fan Zou Lu Bai

Methods 2018 Dec 29. Epub 2018 Dec 29.

Department of Physics, The Pennsylvania State University, University Park, PA 16802, United States; Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, United States; Center for Eukaryotic Gene Regulation, The Pennsylvania State University, University Park, PA 16802, United States. Electronic address:

Time-lapse fluorescence microscopy is a powerful tool to study gene regulation. By probing fluorescent signals in single cells over extended period of time, this method can be used to study the dynamics, noise, movement, memory, inheritance, and coordination, of gene expression during cell growth, development, and differentiation. In combination with a flow-cell device, it can also measure gene regulation by external stimuli. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S10462023183031
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http://dx.doi.org/10.1016/j.ymeth.2018.12.010DOI Listing
December 2018
2 Reads

Manipulating the mechanics of extracellular matrix to study effects on the nucleus and its structure.

Methods 2018 Dec 26. Epub 2018 Dec 26.

Molecular & Cell Biophysics Lab, University of Pennsylvania, Philadelphia, PA 19104, USA; Physical Sciences Oncology Center at Penn, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Tissues such as brain, muscle, and bone differ greatly not only in their biological functions but also in their mechanical properties. Brain is far softer than muscle while bone is the stiffest tissue. Stiffness of extracellular microenvironments affects fundamental cell biological processes such as polarization and DNA replication, which affect nuclear size, shape, and levels of nuclear proteins such as the lamins that modulate gene expression. Read More

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http://dx.doi.org/10.1016/j.ymeth.2018.12.009DOI Listing
December 2018

Selective profiling of ribosomes associated with yeast Upf proteins.

Methods 2018 Dec 26. Epub 2018 Dec 26.

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA 01655-0122, United States. Electronic address:

Ribosomes associated with nonsense-mediated decay factors Upf1, Upf2, or Upf3 were purified by immunoprecipitation, and enrichment and stoichiometry of Upf factors and ribosomal proteins were analyzed by western blot and mass spectrometry. Using a small RNA library preparation protocol that eliminates in-gel RNA and cDNA size selection and incorporates four random nucleotides on each side of the ribosome-protected RNA fragment allowed recovery, detection, and analysis of all size classes of protected fragments from a sample simultaneously. Read More

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http://dx.doi.org/10.1016/j.ymeth.2018.12.008DOI Listing
December 2018
1 Read

Transcriptome-wide identification of A-to-I RNA editing sites using ICE-seq.

Methods 2018 Dec 20. Epub 2018 Dec 20.

Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. Electronic address:

In A-to-I RNA editing, adenosine is converted to inosine in double-stranded regions of RNAs. Inosine, an abundant epitranscriptomic mark, contributes to a wide range of biological processes by regulating gene expression post-transcriptionally. To understand the effect of A-to-I RNA editing on regulation of the epitranscriptome, accurate mapping of inosines is necessary. Read More

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http://dx.doi.org/10.1016/j.ymeth.2018.12.007DOI Listing
December 2018

A lentivirus-based system for Cas9/gRNA expression and subsequent removal by Cre-mediated recombination.

Methods 2018 Dec 19. Epub 2018 Dec 19.

Department of Biochemistry, Molecular Biology and Biophysics, Masonic Cancer Center, Institute for Molecular Virology, Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA; Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address:

A major concern of CRISPR and related genome engineering technologies is off-target mutagenesis from prolonged exposure to Cas9 and related editing enzymes. To help mitigate this concern we added a loxP site to the 3'-LTR of an HIV-based lentiviral vector capable of expressing Cas9/gRNA complexes in a wide variety of mammalian cell types. Transduction of susceptible target cells yields an integrated provirus that expresses the desired Cas9/gRNA complex. Read More

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http://dx.doi.org/10.1016/j.ymeth.2018.12.006DOI Listing
December 2018
1 Read

Defining nonsense-mediated mRNA decay intermediates in human cells.

Methods 2018 Dec 19. Epub 2018 Dec 19.

Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA; Center for RNA Biology, University of Rochester, Rochester, NY 14642, USA. Electronic address:

Nonsense-mediated mRNA decay (NMD) is a cellular mRNA degradation mechanism that inhibits the expression of aberrant mRNAs harboring premature termination codons (PTCs). Recent progress in transcriptome-wide sequencing techniques has revealed that NMD also degrades approximately 5-30% of non-mutated cellular mRNAs in a way that can be regulated in response to various cellular signals. In mammals, NMD is governed by the central NMD factor UPF1, which is activated by phosphorylation after translation terminates at a nonsense codon that triggers NMD. Read More

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http://dx.doi.org/10.1016/j.ymeth.2018.12.005DOI Listing
December 2018
1 Read

Global analysis of RNA metabolism using bio-orthogonal labeling coupled with next-generation RNA sequencing.

Methods 2018 Dec 6. Epub 2018 Dec 6.

Department of Biological Chemistry and Department of Computational Medicine & Bioinformatics, University of Michigan, Room5301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109, USA. Electronic address:

Many open questions in RNA biology relate to the kinetics of gene expression and the impact of RNA binding regulatory factors on processing or decay rates of particular transcripts. Steady state measurements of RNA abundance obtained from RNA-seq approaches are not able to separate the effects of transcription from those of RNA decay in the overall abundance of any given transcript, instead only giving information on the (presumed steady-state) abundances of transcripts. Through the combination of metabolic labeling and high-throughput sequencing, several groups have been able to measure both transcription rates and decay rates of the entire transcriptome of an organism in a single experiment. Read More

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http://dx.doi.org/10.1016/j.ymeth.2018.12.001DOI Listing
December 2018

Practical considerations on performing and analyzing CLIP-seq experiments to identify transcriptomic-wide RNA-protein interactions.

Methods 2018 Dec 6. Epub 2018 Dec 6.

Department of Computer Science, University of Central Florida, Orlando, FL 32816, USA. Electronic address:

RNA-binding proteins are important players in post-transcriptional regulation, such as modulating mRNA splicing, translation, and degradation under diverse biological settings. Identifying and characterizing the RNA substrates is a critical step in deciphering the function and molecular mechanisms of the target RNA-binding proteins. High-throughput sequencing of the RNA fragments isolated by crosslinking immunoprecipitation (CLIP-seq) is one of the standard techniques to identify the in vivo transcriptome-wide binding sites of the target RNA-binding protein. Read More

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http://dx.doi.org/10.1016/j.ymeth.2018.12.002DOI Listing
December 2018

The age of data analytics: converting biomedical data into actionable insights.

Methods 2018 Dec;151:1-2

GLAM - Group on Language, Audio & Music, Imperial College London, SW7 2AZ, London; Chair of Embedded Intelligence for Health Care and Wellbeing, University of Augsburg, 86159, Augsburg, Germany.

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http://dx.doi.org/10.1016/j.ymeth.2018.10.017DOI Listing
December 2018

Engineered viral RNA decay intermediates to assess XRN1-mediated decay.

Methods 2018 Dec 3. Epub 2018 Dec 3.

Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80525, United States; Program in Cell & Molecular Biology, Colorado State University, Fort Collins, CO 80525, United States. Electronic address:

Both RNA synthesis and decay must be balanced within a cell to achieve proper gene expression. Additionally, modulation of RNA decay specifically offers the cell an opportunity to rapidly reshape the transcriptome in response to specific stimuli or cues. Therefore, it is critical to understand the underlying mechanisms through which RNA decay contribute to gene expression homeostasis. Read More

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http://dx.doi.org/10.1016/j.ymeth.2018.11.019DOI Listing
December 2018
4 Reads

Sequencing-based methods for detection and quantitation of ribose methylations in RNA.

Methods 2018 Nov 29. Epub 2018 Nov 29.

Department of Cellular and Molecular Medicine, University of Copenhagen, Denmark. Electronic address:

Ribose methylation is one of the most abundant RNA modifications and is found in all domains of life and all major classes of RNA (rRNA, tRNA, and mRNA). Ribose methylations are introduced by stand-alone enzymes or by generic enzymes guided to the target by small RNA guides. Recent years have seen the development of several sequencing-based methods for RNA modifications relying on different principles. Read More

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http://dx.doi.org/10.1016/j.ymeth.2018.11.017DOI Listing
November 2018

Genome-wide probing RNA structure with the modified DMS-MaPseq in Arabidopsis.

Methods 2018 Nov 29. Epub 2018 Nov 29.

Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA; Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, 77843, USA. Electronic address:

Transcripts have intrinsic propensity to form stable secondary structure that is fundamental to regulate RNA transcription, splicing, translation, RNA localization and turnover. Numerous methods that integrate chemical reactions with next-generation sequencing (NGS) have been applied to study in vivo RNA structure, providing new insights into RNA biology. Dimethyl sulfate (DMS) probing coupled with mutational profiling through NGS (DMS-MaPseq) is a newly developed method for revealing genome-wide or target-specific RNA structure. Read More

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http://dx.doi.org/10.1016/j.ymeth.2018.11.018DOI Listing
November 2018

Current strategies for Site-Directed RNA Editing using ADARs.

Methods 2018 Nov 29. Epub 2018 Nov 29.

The Eugene Bell Center, Marine Biological Laboratory, Woods Hole, MA, USA. Electronic address:

Adenosine Deaminases that Act on RNA (ADARs) are a group of enzymes that catalyze the conversion of adenosines (A's) to inosines (I's) in a process known as RNA editing. Though ADARs can act on different types of RNA, editing events in coding regions of mRNA are of particular interest as I's base pair like guanosines (G's). Thus, every A-to-I change catalyzed by ADAR is read as an A-to-G change during translation, potentially altering protein sequence and function. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S10462023183019
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http://dx.doi.org/10.1016/j.ymeth.2018.11.016DOI Listing
November 2018
6 Reads

Cell-type specific polysome profiling from mammalian tissues.

Methods 2018 Nov 27. Epub 2018 Nov 27.

Department of Biochemistry, University of Illinois, Urbana-Champaign, IL, USA; Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana-Champaign, IL, USA; Cancer Center@ Illinois, University of Illinois, Urbana-Champaign, IL, USA. Electronic address:

The regulation of gene expression occurs through complex relationships between transcription, processing, turnover, and translation, which are only beginning to be elucidated. We know that at least for certain messenger (m) RNAs, processing, modifications, and sequence elements can greatly influence their translational output through recognition by translation and turn-over machinery. Recently, we and others have combined high-throughput sequencing technologies with traditional biochemical methods of studying translation to extend our understanding of these relationships. Read More

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http://dx.doi.org/10.1016/j.ymeth.2018.11.015DOI Listing
November 2018

Combined bead-based multiplex detection of RNA and protein biomarkers: Implications for understanding the time course of skeletal muscle injury and repair.

Methods 2018 Nov 22. Epub 2018 Nov 22.

Applied Physiology Laboratory, University of North Texas, Denton, TX, USA; Department of Biological Sciences, University of North Texas, Denton, TX, USA. Electronic address:

Biological response to skeletal muscle injury time course is generally classified as initial (elevated within first 4-h), delayed (elevated at 24-h), and/or prolonged (elevated at 4-h and sustained to 24-h). Accurate description of this process requires the ability to measure a robust set of RNA and protein biomarkers, yet such an approach is not common and not always feasible. This method proposes a novel experimental approach that focuses on the use of bead-based multiplex detection to measure mRNA, lncRNA, cytokines, soluble cytokine receptors, and myokines at 4-h and 24-h post muscle injury. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S10462023183017
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http://dx.doi.org/10.1016/j.ymeth.2018.11.012DOI Listing
November 2018
6 Reads

Combining single molecule counting with bead-based multiplexing to quantify biological inflammation time course following skeletal muscle injury.

Methods 2018 Nov 22. Epub 2018 Nov 22.

Applied Physiology Laboratory, University of North Texas, Denton, TX, USA; Department of Biological Sciences, University of North Texas, Denton, TX, USA. Electronic address:

Bead-based analysis methods allow for the exploration of a variety of complex biological processes. In particular, these techniques can be applied to better understand how peripheral muscle injury contributes to systemic inflammation. Understanding how these two processes affect one another can give additional insight concerning how changes in inflammation effect readiness to perform in exercise and work environments. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S10462023183020
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http://dx.doi.org/10.1016/j.ymeth.2018.11.013DOI Listing
November 2018
4 Reads

Ancestral transcriptome inference based on RNA-Seq and ChIP-seq data.

Methods 2018 Nov 22. Epub 2018 Nov 22.

Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA. Electronic address:

With the help of high-throughput NGS (next-generation sequencing) technologies, ancestral transcriptome reconstruction is helpful to understand the complexity of transcriptional regulatory systems that underlies the evolution of multiple cellular metazoans with sophisticated functions and distinctive morphologies. To this end, we report a new method of ancestral state inference. The new method used Ornstein-Uhlenbeck (OU) model, which is more biologically realistic, to replace the Brownian motion (BM) model and is suitable for multi-transcriptome data. Read More

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http://dx.doi.org/10.1016/j.ymeth.2018.11.010DOI Listing
November 2018
9 Reads