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: 1108, 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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    A One-enzyme RT-qPCR Assay for SARS-CoV-2, and Procedures for Reagent Production
    Authors:  Sanchita Bhadra, Andre C. Maranhao, Inyup Paik and Andrew D. Ellington, date: 01/20/2021, view: 1087, Q&A: 1
    [Abstract]

    Given the scale of the ongoing COVID-19 pandemic, the need for reliable, scalable testing, and the likelihood of reagent shortages, especially in resource-poor settings, we have developed an RT-qPCR assay that relies on an alternative to conventional viral reverse transcriptases, a thermostable reverse transcriptase/DNA polymerase (RTX) (Ellefson

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    Single-cell qPCR Assay with Massively Parallel Microfluidic System
    Authors:  Marta Prieto-Vila, Takahiro Ochiya and Yusuke Yamamoto, date: 03/20/2020, view: 2415, 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 ...
    Purification of Total RNA from DSS-treated Murine Tissue via Lithium Chloride Precipitation
    Authors:  Emilie Viennois, Anika Tahsin and Didier Merlin, date: 05/05/2018, view: 5558, Q&A: 0
    [Abstract] We have developed a protocol to purify RNA from DSS (Dextran Sulfate Sodium)-treated mouse tissues. This method, which prevents downstream inhibition of q-RT-PCR observed in DSS-treated tissues, relies on successive precipitations with lithium chloride.
    Assessing the Efficacy of Small Molecule Inhibitors in a Mouse Model of Persistent Norovirus Infection
    Authors:  Jana Van Dycke, Johan Neyts and Joana Rocha-Pereira, date: 05/05/2018, view: 4395, Q&A: 0
    [Abstract] Human norovirus is the most common cause of acute gastroenteritis worldwide, resulting in estimated mortality of ~210,000 each year, of whom most are children under the age of five. However, norovirus can infect people of all age groups. There is a risk of prolonged infection in children, the elderly and patients who are immunocompromised. To ...
    Detection and Analysis of Circular RNAs by RT-PCR
    Authors:  Amaresh C Panda and Myriam Gorospe, date: 03/20/2018, view: 14331, Q&A: 4
    [Abstract] Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory ...
    Antisense Oligonucleotide-mediated Knockdown in Mammary Tumor Organoids
    Authors:  Sarah D. Diermeier and David L. Spector, date: 08/20/2017, view: 7382, Q&A: 0
    [Abstract] Primary mammary tumor organoids grown in 3D are an excellent system to study tumor biology. They resemble the organization and physiology of native epithelia more closely than cancer cell lines grown in 2D, and additionally model interactions with the ECM (Boj et al., 2015; Clevers, 2016; Shamir and Ewald, 2014). Mammary tumor organoids ...
    Measurement of 2-methylthio Modifications in Mitochondrial Transfer RNAs by Reverse-transcription Quantitative PCR
    Authors:  Fan-Yan Wei and Kazuhito Tomizawa, date: 01/05/2016, view: 6800, Q&A: 0
    [Abstract] 2-Methylthio-N6-isopentenyladenosine (ms2i6A) is an evolutionally conserved posttranscriptional modification found at position 37 of four mammalian mitochondrial tRNAs, mt-tRNASer(UCN), mt-tRNATrp, mt-tRNAPhe and mt-tRNATyr. The ms2 modification in ms2 ...
    Circular RT-PCR Assay Using Arabidopsis Samples
    Authors:  Runlai Hang, Xian Deng, Chunyan Liu, Beixin Mo and Xiaofeng Cao, date: 07/20/2015, view: 13651, Q&A: 0
    [Abstract] Post-transcriptional processing is critical for RNA biogenesis, in which conventional functional RNA transcripts are generated, such as messenger RNAs (mRNAs), transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) for translation as well as emerging non-coding RNAs with known or unknown regulatory functions. To determine the precise termini of an RNA ...
    RNA-binding Protein Immunoprecipitation (RIP) to Examine AUF1 Binding to Senescence-Associated Secretory Phenotype (SASP) Factor mRNA
    Authors:  Elise Alspach and Sheila A. Stewart, date: 05/20/2015, view: 12158, Q&A: 0
    [Abstract] Immunoprecipitation and subsequent isolation of nucleic acids allows for the investigation of protein:nucleic acid interactions. RNA-binding protein immunoprecipitation (RIP) is used for the analysis of protein interactions with mRNA. Combining RIP with quantitative real-time PCR (qRT-PCR) further enhances the RIP technique by allowing for the ...



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