Protocols in Current Issue
    Charging State Analysis of Transfer RNA from an α-proteobacterium
    Authors:  Liang Yin and Caroline S. Harwood, date: 12/05/2020, view: 700, Q&A: 0
    [Abstract] Transfer RNA (tRNA) is an essential link between the genetic code and proteins. During the process of translation, tRNA is charged with its cognate amino acid and delivers it to the ribosome, thus serving as a substrate of protein synthesis. To analyze the charging state of a particular tRNA, total RNA is purified and analyzed on an acid-urea gel. ...
    Ribosome Purification from an α-proteobacterium and rRNA Analysis by Northern Blot
    Authors:  Liang Yin and Caroline S. Harwood, date: 12/05/2020, view: 717, Q&A: 0
    [Abstract] Ribosomes are an integral part of cellular life. They are complex molecular machines consisting of multiple ribosomal proteins and RNAs. To study different aspects of ribosome composition, many methods have been developed over the decades. Here, we describe how to purify ribosomes from the α-proteobacterium Rhodopseudomonas palustris ...
    Viral Double-Stranded RNA Detection by DNase I and Nuclease S1 digestions in Leishmania parasites
    Authors:  Nathalie Isorce and Nicolas Fasel, date: 05/05/2020, view: 1562, Q&A: 0
    [Abstract] Many RNA viruses are found in protozoan parasites. They can be responsible for more serious pathology or treatment failure. For the detection of viral double-stranded RNA (dsRNA), sequence-dependent and -independent methods are available, such as quantitative real-time PCR and immunofluorescence, dot blot, ELISA or sequencing. The technique ...
    Quantification of Queuosine Modification Levels in tRNA from Human Cells Using APB Gel and Northern Blot
    Authors:  Zaneta Matuszek and Tao Pan, date: 03/20/2019, view: 3782, Q&A: 0
    [Abstract] Queuosine (Q) is a hypermodified base in the wobble anticodon position of tRNAs coding for the amino acids Tyr, His, Asn, and Asp. tRNA Q-modification is introduced by a queuine tRNA-ribosyltransferase (TGT) that replaces the guanine base at G34 at these tRNAs with the modified base. tRNA Q-modification is widely distributed among prokaryotic and ...
    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: 5086, Q&A: 2
    [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 ...
    Visualization of RNA 3’ ends in Escherichia coli Using 3’ RACE Combined with Primer Extension
    Authors:  Xun Wang, Heung Jin Jeon, Monford Paul Abishek N, Jin He and Heon M. Lim, date: 03/05/2018, view: 5532, Q&A: 0
    [Abstract] In this assay, 3’ RACE (Rapid Amplification of cDNA 3’ Ends) followed by PE (primer extension), abbreviated as 3’ RACE-PE is used to identify the mRNA 3’ ends. The following protocol describes the amplification of the mRNA 3’ ends at the galactose operon in E. coli and the corresponding visualization of the PCR products through PE. In PE, ...
    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: 6060, 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.
    RNA Capping by Transcription Initiation with Non-canonical Initiating Nucleotides (NCINs): Determination of Relative Efficiencies of Transcription Initiation with NCINs and NTPs
    Authors:  Jeremy G. Bird, Bryce E. Nickels and Richard H. Ebright, date: 06/20/2017, view: 6344, Q&A: 0
    [Abstract] It recently has been established that adenine-containing cofactors, including nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), and 3’-desphospho-coenzyme A (dpCoA), can serve as ‘non-canonical initiating nucleotides’ (NCINs) for transcription initiation by bacterial and eukaryotic cellular RNA ...
    RNA-dependent RNA Polymerase Assay for Hepatitis E Virus
    Authors:  Vidya P. Nair, Saumya Anang, Akriti Srivastava and Milan Surjit, date: 04/05/2017, view: 7936, Q&A: 0
    [Abstract] RNA-dependent RNA polymerase (RdRp) is essential for the replication of viral RNA for RNA viruses. It synthesizes the complementary strand of viral genomic RNA, which is used subsequently as a template to generate more copies of viral genome. This assay measures activity of the hepatitis E virus (HEV) RdRp. In contrast to protocols available to ...
    RNA Strand Displacement Assay for Hepatitis E Virus Helicase
    Authors:  Vidya P. Nair and Milan Surjit, date: 04/05/2017, view: 6134, Q&A: 0
    [Abstract] The hepatitis E virus (HEV) helicase uses ATP to unwind the RNA duplexes. This is an essential step for viral replication. This protocol aims to measure the double strand RNA unwinding activity of the HEV helicase.

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