Molecular Biology

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    Protocols in Current Issue
    Optogenetic Inactivation of Transcription Factors in the Early Embryo of Drosophila
    Authors:  Stephen L. McDaniel and Melissa M. Harrison, date: 07/05/2019, view: 236, Q&A: 0
    [Abstract] The early embryo of Drosophila melanogaster exists as a rapidly dividing syncytium of nuclei that are transcriptionally silent. Maternally deposited factors are required to awaken the genome and assist in the transition from maternal to zygotic control of development. Because many of these essential factors are maternally deposited and ...
    An Efficient Approach for RNA Extraction from Boar Sperm and Seminal Plasma
    Authors:  Vanmathy R. Kasimanickam and Ramanathan K. Kasimanickam, date: 07/05/2019, view: 180, Q&A: 0
    [Abstract] Despite transcriptional silencing in mature sperm and cytoplasmic expulsion of RNA during the final sperm maturation process, thousands of RNAs have been successfully identified in ejaculated sperm. Although most of RNAs’ function is still unknown, it is suggested that sperm RNAs have a vital biological role in fertilization and post-fertilization ...
    Immunoprecipitation and Sequencing of Acetylated RNA
    Authors:  Daniel Arango, David Sturgill and Shalini Oberdoerffer, date: 06/20/2019, view: 534, Q&A: 0
    [Abstract] Generation of the epitranscriptome through chemical modifications of protein-coding messenger RNAs (mRNAs) has emerged as a new mechanism of post-transcriptional gene regulation. While most mRNA modifications are methylation events, a single acetylated ribonucleoside has been described in eukaryotes, occurring at the N4-position of cytidine ...
    Organelle-associated rRNA Degradation
    Authors:  Jinliang Huang and Geng Wang, date: 06/05/2019, view: 321, Q&A: 0
    [Abstract] Cytosolic rRNAs are highly dynamic and can be degraded under conditions such as apoptosis, starvation and magnesium depletion. The degradation is also related to their specific localization, as fractions of cytosolic ribosomes are localized on the surfaces of intracellular organelles, such as endoplasmic reticulum (ER) and mitochondria. Such ...
    Improved Mammalian Mitochondrial RNA Isolation
    Authors:  Jinliang Huang and Geng Wang, date: 06/05/2019, view: 418, Q&A: 0
    [Abstract] Mitochondria have two sets of RNAs. One is encoded in mitochondrial genome, and the other that consists of imported RNAs within mitochondria and cytosolic RNAs associated with mitochondrial outer membrane is encoded in the nucleus. These mitochondrial RNAs play important roles in mitochondrion biosynthesis and signaling in and out of mitochondria. ...
    Minigene Assay to Evaluate CRISPR/Cas9-based Excision of Intronic Mutations that Cause Aberrant Splicing in Human Cells
    Authors:  David J. Sanz and Patrick T. Harrison, date: 06/05/2019, view: 284, Q&A: 0
    [Abstract] The construction of Hybrid minigenes provides a robust and simple strategy to study the effects of disease-causing mutations on mRNA splicing when biological material from patient cells is not available. Hybrid minigenes can be used as splicing reporter plasmids allow RNA expression and heterologous splicing reactions between synthetic splicing ...
    Enterovirus Competition Assay to Assess Replication Fitness
    Authors:  Valeria Lulla and Andrew E. Firth, date: 05/20/2019, view: 382, Q&A: 0
    [Abstract] In virology the difference between the fitness of two viruses can be determined by using various methods, such as virus titer, growth curve analysis, measurement of virus infectivity, analysis of produced RNA copies and viral protein production. However, for closely performing viruses, it is often very hard to distinguish the differences. In vitro ...
    Northern Blot with IR Fluorescent Probes: Strategies for Probe Preparation
    Authors:  Christopher Fields, Peike Sheng, Bret Miller, Tianqi Wei and Mingyi Xie, date: 04/20/2019, view: 807, Q&A: 0
    [Abstract] Northern blot is a molecular biology technique that can detect, quantify, and determine the molecular weight of RNA. Recently, we published a protocol utilizing near-infrared (IR) fluorescent probes in Northern blot (irNorthern). Our method is as sensitive as other non-radioactive methods but is more straightforward and versatile. Additionally, we ...
    Identification of RNase-sensitive LINE-1 Ribonucleoprotein Interactions by Differential Affinity Immobilization
    Authors:  Hua Jiang, Martin S. Taylor, Kelly R. Molloy, Ilya Altukhov and John LaCava, date: 04/05/2019, view: 865, Q&A: 0
    [Abstract] Long Interspersed Nuclear Element-1 (LINE-1, L1) constitutes a family of autonomous, self-replicating genetic elements known as retrotransposons. Although most are inactive, copious L1 sequences populate the human genome. L1s proliferate in a ‘copy-and-paste’ fashion through an RNA intermediate; a full-length L1 transcript is ~6,000 nucleotides ...
    Preparation of RNA 3’ End Sequencing Libraries of Total and 4-thiouracil Labeled RNA for Simultaneous Measurement of Transcription, RNA Synthesis and Decay in S. cerevisiae
    Authors:  Manfred Schmid, Agnieszka Tudek and Torben Heick Jensen, date: 03/20/2019, view: 754, Q&A: 0
    [Abstract] Cellular RNA levels are determined by the rates of RNA transcription from the gene template and subsequent RNA stability. Knowledge about both transcription and RNA decay is, therefore, necessary to interpret RNA levels and gene expression, especially during cellular processes where these parameters change. Numerous experimental strategies have ...



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