Molecular Biology

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    Protocols in Current Issue
    Analysis of Pseudomonas aeruginosa c-di-GMP High and Low Subpopulations Using Flow-assisted Cell Sorting (FACS) and Quantitative Reverse Transcriptase PCR (qRT-PCR)
    Authors:  Catherine R. Armbruster and Matthew R. Parsek, date: 01/20/2021, view: 167, Q&A: 0
    [Abstract]

    Cyclic diguanylate monophosphate (c-di-GMP) is a second messenger signaling molecule that drives the transition from planktonic to the biofilm mode of growth in many bacterial species. Pseudomonas aeruginosa has at least two surface sensing systems that produce c-di-GMP in response to surface attachment, the Wsp and Pil-Chp systems. We recently

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    RNA ImmunoGenic Assay: A Method to Detect Immunogenicity of in vitro Transcribed mRNA in Human Whole Blood
    [Abstract] The mRNA therapeutics is a new class of medicine to treat many various diseases. However, in vitro transcribed (IVT) mRNA triggers immune responses due to recognition by human endosomal and cytoplasmic RNA sensors, but incorporation of modified nucleosides have been shown to reduce such responses. Therefore, an assay signifying important ...
    Charging State Analysis of Transfer RNA from an α-proteobacterium
    Authors:  Liang Yin and Caroline S. Harwood, date: 12/05/2020, view: 430, 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. ...
    Candida albicans Culture, Cell Harvesting, and Total RNA Extraction
    Authors:  Max V. Cravener and Aaron P. Mitchell, date: 11/05/2020, view: 687, Q&A: 0
    [Abstract]

    Transcriptional analysis has become a cornerstone of biological research, and with the advent of cheaper and more efficient sequencing technology over the last decade, there exists a need for high-yield and efficient RNA extraction techniques. Fungi such as the human pathogen Candida albicans present a unique obstacle to RNA purification in the

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    Colorimetric RT-LAMP Methods to Detect Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)
    [Abstract] Standard diagnostic methods of Coronavirus Disease 2019 (COVID-19) rely on RT-qPCR technique which have limited point-of-care test (POCT) potential due to necessity of dedicated equipment and specialized personnel. LAMP, an isothermal nucleic acid amplification test (NAAT), is a promising technique that may substitute RT-qPCR for POCT of genomic ...
    Advances in Proximity Ligation in situ Hybridization (PLISH)
    Authors:  Monica Nagendran, Adam M Andruska, Pehr B Harbury and Tushar J Desai, date: 11/05/2020, view: 1052, Q&A: 0
    [Abstract] Understanding tissues in the context of development, maintenance and disease requires determining the molecular profiles of individual cells within their native in vivo spatial context. We developed a Proximity Ligation in situ Hybridization technology (PLISH) that enables quantitative measurement of single cell gene expression ...
    A Protocol for Simple, Rapid, and Direct Detection of SARS-CoV-2 from clinical samples, using Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP)
    [Abstract] SARS-CoV-2 has quickly spread all around the globe causing illness and wide damages. Most countries were unprepared for such a rapid spread and crisis. This led to various strategies for effective control of the new pandemic. A key aspect in all countries was to effectively test the population for the virus. Most countries chose a lockdown ...
    Analyzing (Re)Capping of mRNA Using Transcript Specific 5' End Sequencing
    Authors:  Daniel del Valle Morales and Daniel R. Schoenberg, date: 10/20/2020, view: 534, Q&A: 0
    [Abstract] The 5′ cap is a ubiquitous feature of eukaryotic mRNAs. It is added in the nucleus onto newly synthesized pre-mRNA, and in the cytoplasm onto mRNAs after decapping or endonuclease cleavage. Cytoplasmic recapping can occur after loss of the cap at the native 5′ end, or downstream within the body of the mRNA. The identification and location of ...
    Using RNA Sequencing and Spike-in RNAs to Measure Intracellular Abundance of lncRNAs and mRNAs
    Authors:  Megan D. Schertzer, McKenzie M. Murvin and J. Mauro Calabrese, date: 10/05/2020, view: 769, Q&A: 0
    [Abstract] Long noncoding RNAs (lncRNAs) play essential roles in normal physiology and in disease but their mechanisms of action can be challenging to identify. For mechanistic studies, it is often useful to know a lncRNA’s intracellular abundance, i.e., approximately how many molecules of the lncRNA are present in a typical cell of a cell-type of ...
    Nonenzymatic RNA-templated Synthesis of N3′→P5′ Phosphoramidate DNA
    Authors:  Derek K. O'Flaherty, Lijun Zhou and Jack W. Szostak, date: 09/05/2020, view: 1500, Q&A: 0
    [Abstract] The RNA world hypothesis describes a scenario where early life forms relied on RNA to govern both inheritance and catalyze useful chemical reactions. Prior to the emergence of enzymes capable of replicating the RNA genome, a nonenzymatic replication process would have been necessary to initiate Darwinian Evolution. However, the one-pot ...



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