Mammalia

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    Protocols in Current Issue
    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: 1467, 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 ...
    Organelle-associated rRNA Degradation
    Authors:  Jinliang Huang and Geng Wang, date: 06/05/2019, view: 878, 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: 1411, 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. ...
    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: 2617, Q&A: 1
    [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: 1661, 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 ...
    RNA Cap Methyltransferase Activity Assay
    Authors:  Jackson B. Trotman and Daniel R. Schoenberg, date: 03/20/2018, view: 2890, Q&A: 0
    [Abstract] Methyltransferases that methylate the guanine-N7 position of the mRNA 5’ cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing ...
    Immunoprecipitation of Tri-methylated Capped RNA
    Authors:  Karen E. Hayes, Jamie A. Barr, Mingyi Xie, Joan A. Steitz and Ivan Martinez, date: 02/05/2018, view: 4157, Q&A: 0
    [Abstract] Cellular quiescence (also known as G0 arrest) is characterized by reduced DNA replication, increased autophagy, and increased expression of cyclin-dependent kinase p27Kip1. Quiescence is essential for wound healing, organ regeneration, and preventing neoplasia. Previous findings indicate that microRNAs (miRNAs) play an ...
    Protocol for Establishing a Multiplex Image-based Autophagy RNAi Screen in Cell Cultures
    Authors:  Jennifer Jung and Christian Behrends, date: 09/05/2017, view: 4737, Q&A: 0
    [Abstract] Autophagy is a recycling pathway, in which intracellular cargoes including protein aggregates and bacteria are engulfed by autophagosomes and subsequently degraded after fusion with lysosomes. Dysregulation of this process is involved in several human diseases such as cancer or neurodegeneration. Hence, advancing our understanding of how autophagy ...
    RNA Interference Screening to Identify Proliferation Determinants in Breast Cancer Cells
    [Abstract] RNAi screening technology has revealed unknown determinants of various biological signaling pathways in biomedical studies. This protocol provided detailed information about how to use RNAi screening to identify proliferation determinants in breast tumor cells. siRNA-based libraries targeting against Estrogen receptor (ER)-network, including 631 ...
    Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System
    Authors:  Reka A. Haraszti, Andrew Coles, Neil Aronin, Anastasia Khvorova and Marie-Cécile Didiot, date: 06/20/2017, view: 4919, Q&A: 0
    [Abstract] Efficient delivery of oligonucleotide therapeutics, i.e., siRNAs, to the central nervous system represents a significant barrier to their clinical advancement for the treatment of neurological disorders. Small, endogenous extracellular vesicles were shown to be able to transport lipids, proteins and RNA between cells, including neurons. ...



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