Microbiology

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    Protocols in Current Issue
    Detection and Differentiation of Multiple Viral RNAs Using Branched DNA FISH Coupled to Confocal Microscopy and Flow Cytometry
    Authors:  Nicholas van Buuren and Karla Kirkegaard, date: 10/20/2018, view: 1699, Q&A: 0
    [Abstract] Due to the exceptionally high mutation rates of RNA-dependent RNA polymerases, infectious RNA viruses generate extensive sequence diversity, leading to some of the lowest barriers to the development of antiviral drug resistance in the microbial world. We have previously discovered that higher barriers to the development of drug resistance can be ...
    A Small RNA Isolation and Sequencing Protocol and Its Application to Assay CRISPR RNA Biogenesis in Bacteria
    [Abstract] Next generation high-throughput sequencing has enabled sensitive and unambiguous analysis of RNA populations in cells. Here, we describe a method for isolation and strand-specific sequencing of small RNA pools from bacteria that can be multiplexed to accommodate multiple biological samples in a single experiment. Small RNAs are isolated by ...
    Single Genome Sequencing of Expressed and Proviral HIV-1 Envelope Glycoprotein 120 (gp120) and nef Genes
    [Abstract] The current study provides detailed protocols utilized to amplify the complete HIV-1 gp120 and nef genes from single copies of expressed or integrated HIV present in fresh-frozen autopsy tissues of patients who died while on combined antiretroviral therapy (cART) with no detectable plasma viral load (pVL) at death (Lamers et al ...
    Ultradeep Pyrosequencing of Hepatitis C Virus to Define Evolutionary Phenotypes
    [Abstract] Analysis of hypervariable regions (HVR) using pyrosequencing techniques is hampered by the ability of error correction algorithms to account for the heterogeneity of the variants present. Analysis of between-sample fluctuations to virome sub-populations, and detection of low frequency variants, are unreliable through the application of arbitrary ...
    Sequencing of Ebola Virus Genomes Using Nanopore Technology
    Author:  Thomas Hoenen, date: 11/05/2016, view: 6276, Q&A: 0
    [Abstract] Sequencing of virus genomes during disease outbreaks can provide valuable information for diagnostics, epidemiology, and evaluation of potential countermeasures. However, particularly in remote areas logistical and technical challenges can be significant. Nanopore sequencing provides an alternative to classical Sanger and next-generation ...
    Purification of Bacterial RNA from Infected Macrophages
    Authors:  Lior Lobel, Nadejda Sigal, Anna Pasechnek and Anat A. Herskovits, date: 11/20/2015, view: 6348, Q&A: 0
    [Abstract] Studying the transcriptome of bacterial pathogens during infection is a very informative and effective tool for discovering genes that contribute to successful infection. However, isolating bacterial RNA from infected cells or tissues is a challenging process due to the much higher amounts of host RNA in the lysates of infected cells. We have ...
    Quantification of Total and 2-LTR (Long terminal repeat) HIV DNA, HIV RNA and Herpesvirus DNA in PBMCs
    Authors:  Marta Massanella, Sara Gianella, Steven M. Lada, Douglas D. Richman and Matthew C. Strain, date: 06/05/2015, view: 7709, Q&A: 0
    [Abstract] Almost all individuals infected with human immunodeficiency virus (HIV) are also infected with cytomegalovirus (CMV) and Epstein Barr virus (EBV). The aims of our studies have included characterizing and measuring the latent HIV reservoir and understanding the association between asymptomatic replication of CMV (and other herpesvirus, including ...
    Quantification of HIV RNA and Human Herpesvirus DNA in Seminal Plasma
    Authors:  Milenka V. Vargas-Meneses, Marta Massanella, Caroline C. Ignacio and Sara Gianella, date: 05/05/2015, view: 5946, Q&A: 0
    [Abstract] Multiple viruses can co-infect the genital tract, modifying the immunologic and virologic milieu and possibly playing a role in viral transmission and pathogenesis. The aim of our studies has been to understand the complex relationships between HIV-1 RNA, and multiple human herpesviruses known to frequently replicate in the genital tract of ...
    RNA Isolation from Synechocystis
    Authors:  Kirill S. Mironov and Dmitry A. Los, date: 03/20/2015, view: 14461, Q&A: 2
    [Abstract] The protocol describes the procedure of total RNA isolation from cells of the cyanobacterium Synechocystis sp. PCC 6803. This protocol is also applicable to Synechococcus elongatus PCC 7942 and PCC 6301, Thermosynechococcus vulcanus, and other unicellular and filamentous species of cyanobacteria that do not have thick ...
    In vitro Transcription (IVT) and tRNA Binding Assay
    Author:  Sonja MK Schoenfelder, date: 09/20/2014, view: 8705, Q&A: 0
    [Abstract] This protocol describes the coupling of (i) “live” in vitro RNA transcription with (ii) binding by a radiolabeled, pre-formed tRNA followed by native gel electrophoresis and phosphorimager scan to visualize the complex. The necessity arose from the stable structure that one RNA forms in the absence of its interaction partner. The T-box ...



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