Molecular Biology

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    Protocols in Current Issue
    Purification of Mitochondrial Ribosomal Complexes from Trypanosoma cruzi and Leishmania tarentolae for Cryo-EM Analysis
    Authors:  Stéphanie Durrieu-Gaillard, Marie Sissler and Yaser Hashem, date: 05/20/2022, view: 21, Q&A: 0
    [Abstract]

    Kinetoplastids are unicellular eukaryotic parasites responsible for human pathologies such as Chagas disease, sleeping sickness or Leishmaniasis, caused by Trypanosoma cruzi, Trypanosoma brucei, and various Leishmania spp., respectively. They harbor a single large mitochondrion that is essential for the survival of the parasite. Interestingly,

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    circFL-seq, A Full-length circRNA Sequencing Method
    Authors:  Zelin Liu and Ence Yang, date: 04/20/2022, view: 775, Q&A: 1
    [Abstract]

    Due to overlapping sequences with linear cognates, identifying internal sequences of circular RNA (circRNA) remains a challenge. Recently, we have developed a full-length circRNA sequencing method (circFL-seq) and computational pipeline, to profile ordinary and fusion circRNA at the isoform level. Compared to short-read RNA-seq, rolling circular

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    RNA Interference Method for Gene Function Analysis in the Japanese Rhinoceros Beetle Trypoxylus dichotomus
    Authors:  Kazuki Sakura, Shinichi Morita and Teruyuki Niimi, date: 04/20/2022, view: 704, Q&A: 0
    [Abstract]

    In the Japanese rhinoceros beetle Trypoxylus dichotomus, various candidate genes required for a specific phenotype of interest are listed by next-generation sequencing analysis. Their functions were investigated using RNA interference (RNAi) method, the only gene function analysis tool for T. dichotomus developed so far. The summarized procedure

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    Plasmid and Sequencing Library Preparation for CRISPRi Barcoded Expression Reporter Sequencing (CiBER-seq) in Saccharomyces cerevisiae
    Authors:  Ryan Y Muller, Zuriah A Meacham and Nicholas T Ingolia, date: 04/05/2022, view: 679, Q&A: 0
    [Abstract]

    Genetic networks regulate nearly all biological processes, including cellular differentiation, homeostasis, and immune responses. Determining the precise role of each gene within a regulatory network can explain its overall, integrated function, and pinpoint mechanisms underlying misregulation in disease states. Transcriptional reporter assays are

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    A Novel PCR-Based Methodology for Viral Detection Utilizing Mechanical Homogenization
    Authors:  Zachary P. Morehouse, Caleb M. Proctor, Gabriella L. Ryan and Rodney J. Nash, date: 03/05/2022, view: 720, Q&A: 0
    [Abstract]

    The impact of viral diseases on human health is becoming increasingly prevalent globally with the burden of disease being shared between resource-rich and poor areas. As seen in the global pandemic caused by SARS-CoV-2, there is a need to establish viral detection techniques applicable to resource-limited areas that provide sensitive and specific

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    An in vitro Assay of mRNA 3’ end Using the E. coli Cell-free Expression System
    Authors:  Monford Paul Abishek N and Heon M. Lim, date: 02/20/2022, view: 1247, Q&A: 0
    [Abstract]

    At the end of about 80% of the operon in Escherichia coli, translation termination decouples transcription, leading to Rho-dependent transcription termination (RDT). However, no in vitro or in vivo assay system has proven to be good enough to see the 3’ end of the mRNA generated by RDT. Here, we present a cell-free assay system that

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    Normalized Ribo-Seq for Quantifying Absolute Global and Specific Changes in Translation
    Authors:  Katharina Hoerth, Sonja Reitter and Johanna Schott, date: 02/20/2022, view: 876, Q&A: 0
    [Abstract] Ribosome profiling (Ribo-Seq) is a highly sensitive method to quantify ribosome occupancies along individual mRNAs on a genome-wide scale. Hereby, ribosome-protected fragments (= footprints) are generated by nuclease digestion, isolated, and sequenced together with the corresponding randomly fragmented input samples, to determine ribosome ...
    Pull-down of Biotinylated RNA and Associated Proteins
    Authors:  Jagadeesh K. Uppala, Chandrima Ghosh, Grzegorz Sabat and Madhusudan Dey, date: 02/20/2022, view: 1043, Q&A: 0
    [Abstract]

    Mapping networks of RNA-protein interactions in cells is essential for understanding the inner workings of many biological processes, including RNA processing, trafficking, and translation. Current in vivo methods for studying protein-RNA interactions rely mostly on purification of poly(A) transcripts, which represent only ~2–3% of total

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    Combination of Immunofluorescence and Quantitative Fluorescence In-situ Hybridization for Analysing Differential Gene Expression in the Niche Cells of the Drosophila Lymph Gland
    Authors:  Parvathy Ramesh, Sushmit Ghosh and Lolitika Mandal, date: 01/20/2022, view: 1194, Q&A: 0
    [Abstract]

    The Drosophila larval haematopoietic organ or lymph gland consists of multiple cell types arranged in zones. The smallest stem cell compartment consists of 40-45 cells that constitute the haematopoietic niche. In order to analyse the haematopoietic niche, it needs to be labelled with a specific antibody to differentiate it from the other cell

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    Total Nucleic Acid Extraction from Single Zebrafish Embryos for Genotyping and RNA-seq
    Authors:  Neha Wali, Munise Merteroglu, Richard J. White and Elisabeth M. Busch-Nentwich, date: 01/05/2022, view: 1500, Q&A: 0
    [Abstract]

    RNA sequencing allows for the quantification of the transcriptome of embryos to investigate transcriptional responses to various perturbations (e.g., mutations, infections, drug treatments). Previous protocols either lack the option to genotype individual samples, or are laborious and therefore difficult to do at a large scale. We have developed a

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