Molecular Biology

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    Protocols in Current Issue
    Yeast Single-cell RNA-seq, Cell by Cell and Step by Step
    [Abstract] Single-cell RNA-seq (scRNA-seq) has become an established method for uncovering the intrinsic complexity within populations. Even within seemingly homogenous populations of isogenic yeast cells, there is a high degree of heterogeneity that originates from a compact and pervasively transcribed genome. Research with microorganisms such as yeast ...
    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: 281, 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 ...
    Three-dimensional Reconstruction and Quantification of Proteins and mRNAs at the Single-cell Level in Cultured Cells
    Authors:  Xueer Jiang, Ingrid Brust-Mascher and Li-En Jao, date: 08/20/2019, view: 527, Q&A: 0
    [Abstract] Gene expression is often regulated by the abundance, localization, and translation of mRNAs in both space and time. Being able to visualize mRNAs and protein products in single cells is critical to understand this regulatory process. The development of single-molecule RNA fluorescence in situ hybridization (smFISH) allows the detection of ...
    Cell Type-specific mRNA Purification in Caenorhabditis elegans via Translating Ribosome Affinity Purification
    Authors:  Ian G. McLachlan and Steven W. Flavell, date: 08/05/2019, view: 386, Q&A: 0
    [Abstract] Cell type-specific molecular profiling is widely used to gain new insights into the diverse cell types that make up complex biological tissues. Translating ribosome affinity purification (TRAP) is a method in which the cell type-specific expression of epitope-tagged ribosomal subunits allows one to purify actively translating mRNAs without the ...
    In vitro RNA Cleavage Assays to Characterize IRE1-dependent RNA Decay
    Authors:  G. Elif Karagöz, Jirka Peschek, Peter Walter and Diego Acosta-Alvear, date: 07/20/2019, view: 1000, Q&A: 0
    [Abstract] The kinase/RNase IRE1 is a key effector of the cellular response to endoplasmic reticulum stress. The RNase activity of IRE1 can be measured in cells or in the test tube. Here we describe a protocol for the in vitro cleavage and analysis of RNA substrates of IRE1. The method consists of the in vitro transcription, purification ...
    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: 836, 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 ...
    Total RNA Isolation from Separately Established Monolayer and Hydrogel Cultures of Human Glioblastoma Cell Line
    Authors:  Manasi P Jogalekar and Elba E Serrano, date: 07/20/2019, view: 791, Q&A: 0
    [Abstract] Astrocytoma is an invasive carcinoma occurring in the nervous system and currently lacks effective treatment options. A deeper understanding of the mechanisms of tumorigenesis and tumor progression is needed in order to develop novel therapeutic strategies. Recent advances in in vitro culture systems have demonstrated that the use of ...
    Optogenetic Inactivation of Transcription Factors in the Early Embryo of Drosophila
    Authors:  Stephen L. McDaniel and Melissa M. Harrison, date: 07/05/2019, view: 736, 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: 732, 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: 977, 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 ...



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