Biochemistry

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    Protocols in Current Issue
    Single-cell qPCR Assay with Massively Parallel Microfluidic System
    Authors:  Marta Prieto-Vila, Takahiro Ochiya and Yusuke Yamamoto, date: 03/20/2020, view: 183, Q&A: 0
    [Abstract] The single-cell transcriptome is the set of messenger RNA molecules expressed in one cell. It is extremely variable and changes according to external, physical and biochemical conditions. Due to sensitivity shortages, most of genetic studies use bulk samples, providing only the average gene expression. Single-cell technologies have provided a ...
    Ribonucleoprotein Immunoprecipitation (RIP) Analysis
    Authors:  Jennifer L. Martindale, Myriam Gorospe and Maria L. Idda, date: 01/20/2020, view: 594, Q&A: 0
    [Abstract] RNAs and RNA-binding proteins (RBPs) can interact dynamically in ribonucleoprotein (RNP) complexes that play important roles in controlling gene expression programs. One of the powerful ways to investigate changes in the association of RNAs with an RBP of interest is by immunoprecipitation (IP) analysis of native RNPs. RIP (RNP ...
    Laser Capture Micro-dissection (LCM) of Neonatal Mouse Forebrain for RNA Isolation
    Authors:  Achira Roy, Mei Deng, Kimberly A. Aldinger, Ian A. GLass and Kathleen J. Millen, date: 01/05/2020, view: 613, Q&A: 0
    [Abstract] Precise and reproducible isolation of desired cell types or layers from heterogeneous tissues is crucial to analyze specific gene profiles and molecular interactions in vivo. Forebrain is the core site of higher functions, like cognition and memory consolidation. It is composed of heterogeneous and distinct cell types, interconnected to ...
    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: 1738, 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 ...
    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: 3182, 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 ...
    Preparation of Sequencing RNA Libraries through Chemical Cross-linking Coupled to Affinity Purification (cCLAP) in Saccharomyces cerevisiae
    Authors:  Congwei Wang, Julie Weidner and Anne Spang, date: 10/05/2018, view: 2966, Q&A: 0
    [Abstract] Ribonucleoprotein particles (mRNPs) are complexes consisting of mRNAs and RNA-binding proteins (RBPs) which control mRNA transcription localization, turnover, and translation. Some mRNAs within the mRNPs have been shown to undergo degradation or storage. Those transcripts can lack general mRNA elements, like the poly(A) tail or 5’ cap structure, ...
    Enzymatic Synthesis and Fractionation of Fluorescent PolyU RNAs
    [Abstract] The physical properties of viral-length polyuridine (PolyU) RNAs, which cannot base-pair and form secondary structures, are compared with those of normal-composition RNAs, composed of comparable numbers of each of A, U, G and C nucleobases. In this protocol, we describe how to synthesize fluorescent polyU RNAs using the enzyme polynucleotide ...
    Purification of RNA Mango Tagged Native RNA-protein Complexes from Cellular Extracts Using TO1-Desthiobiotin Fluorophore Ligand
    Authors:  Shanker Shyam Sundhar Panchapakesan, Sunny C. Y. Jeng and Peter J. Unrau, date: 04/05/2018, view: 3535, Q&A: 0
    [Abstract] A native purification strategy using RNA Mango for RNA based purification of RNA-protein complexes is described. The RNA Mango aptamer is first genetically engineered into the RNA of interest. RNA Mango containing complexes obtained from cleared cellular native extracts are then immobilized onto TO1-Desthiobiotin saturated streptavidin agarose ...
    Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation
    Authors:  Soufiane Aboulhouda, Rachael Di Santo, Gabriel Therizols and David Weinberg, date: 10/05/2017, view: 6199, Q&A: 0
    [Abstract] The efficiency with which proteins are produced from mRNA molecules can vary widely across transcripts, cell types, and cellular states. Methods that accurately assay the translational efficiency of mRNAs are critical to gaining a mechanistic understanding of post-transcriptional gene regulation. One way to measure translational efficiency is to ...
    Mapping RNA Sequences that Contact Viral Capsid Proteins in Virions
    Authors:  C. Cheng Kao, Ella Chuang, James Ford, Jie Huang, Ram Podicheti and Doug B. Rusch, date: 07/20/2017, view: 5024, Q&A: 0
    [Abstract] We have adapted the methodology of CLIP-seq (Crosslinking-Immunoprecipitation and DNA Sequencing) to map the segments of encapsidated RNAs that contact the protein shells of virions. Results from the protocol report on the RNA sequences that contact the viral capsid.



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