Molecular Biology

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    Protocols in Current Issue
    Using RNA Sequencing and Spike-in RNAs to Measure Intracellular Abundance of lncRNAs and mRNAs
    Authors:  Megan D. Schertzer, McKenzie M. Murvin and J. Mauro Calabrese, date: 10/05/2020, view: 253, Q&A: 0
    [Abstract] Long noncoding RNAs (lncRNAs) play essential roles in normal physiology and in disease but their mechanisms of action can be challenging to identify. For mechanistic studies, it is often useful to know a lncRNA’s intracellular abundance, i.e., approximately how many molecules of the lncRNA are present in a typical cell of a cell-type of ...
    Low-cost and Multiplexable Whole mRNA-Seq Library Preparation Method with Oligo-dT Magnetic Beads for Illumina Sequencing Platforms
    Authors:  Makoto Kashima, Ayumi Deguchi, Ayumi Tezuka and Atsushi J. Nagano, date: 06/20/2020, view: 1550, Q&A: 0
    [Abstract] RNA-Seq is a powerful method for transcriptome analysis used in varied field of biology. Although several commercial products and hand-made protocols enable us to prepare RNA-Seq library from total RNA, their cost are still expensive. Here, we established a low-cost and multiplexable whole mRNA-Seq library preparation method for illumine ...
    Circadian Gene Profiling in Laser Capture Microdissected Mouse Club Cells
    Authors:  Zhenguang Zhang and Andrew Loudon, date: 04/20/2020, view: 1101, Q&A: 0
    [Abstract] Cell heterogeneity is high in tissues like lung. Research conducted on pure population of cells usually offers more insights than bulk tissues, such as circadian clock work. In this protocol, we provide a detailed work flow on how to do circadian clock study by RNA seq in laser capture micro-dissected mouse lung club cells. The method uses frozen ...
    mRNA Extraction from Gill Tissue for RNA-sequencing
    Authors:  Jukka-Pekka Verta and Felicity Jones, date: 03/05/2020, view: 1339, Q&A: 0
    [Abstract] Adaptation is thought to proceed in part through spatial and temporal changes in gene expression. Fish species such as the threespine stickleback are powerful vertebrate models to study the genetic architecture of adaptive changes in gene expression since divergent adaptation to different environments is common, they are abundant and easy to study ...
    RNA Sequencing of Single Myofibers from Mus musculus
    Authors:  Darren M. Blackburn, Felicia Lazure and Vahab D. Soleimani, date: 02/20/2020, view: 2302, Q&A: 0
    [Abstract] Whole transcriptome analysis is a key method in biology that allows researchers to determine the effect a condition has on gene regulation. One difficulty in RNA sequencing of muscle is that traditional methods are performed on the whole muscle, but this captures non-myogenic cells that are part of the muscle. In order to analyze only the ...
    Structural Alignment and Covariation Analysis of RNA Sequences
    Authors:  Nicolas J. Tourasse and Fabien Darfeuille, date: 02/05/2020, view: 1662, Q&A: 0
    [Abstract] RNA molecules adopt defined structural conformations that are essential to exert their function. During the course of evolution, the structure of a given RNA can be maintained via compensatory base-pair changes that occur among covarying nucleotides in paired regions. Therefore, for comparative, structural, and evolutionary studies of RNA ...
    Application of a Modified Smart-seq2 Sample Preparation Protocol for Rare Cell Full-length Single-cell mRNA Sequencing to Mouse Oocytes
    Authors:  Rebecca S. Treger, Scott D. Pope, Xiaojun Xing and Akiko Iwasaki, date: 08/20/2019, view: 2498, Q&A: 0
    [Abstract] Endogenous retroviruses (ERV) are transposable retroelements that form ~10% of the murine genome and whose family members are differentially expressed throughout embryogenesis. However, precise regulation of ERV in germ cells remains unclear. To investigate ERV expression in oocytes, we adapted a single-cell mRNA-sequencing library preparation ...
    Axon-seq for in Depth Analysis of the RNA Content of Neuronal Processes
    Authors:  Jik Nijssen, Julio Aguila and Eva Hedlund, date: 07/20/2019, view: 5240, Q&A: 0
    [Abstract] Neuronal processes have an RNA composition that is distinct from the cell body. Therefore, to fully understand neuronal biology in health and disease we need to study both somas, dendrites and axons. Here we describe a detailed protocol of a newly refined method, Axon-seq, for RNA sequencing of axons (and dendrites) grown in isolation using single ...
    Enterovirus Competition Assay to Assess Replication Fitness
    Authors:  Valeria Lulla and Andrew E. Firth, date: 05/20/2019, view: 2866, 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 ...
    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: 2430, 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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