Molecular Biology

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    Protocols in Current Issue
    A Highly Sensitive Method to Efficiently Profile the Histone Modifications of FFPE Samples

    The majority of biopsies in both basic research and translational cancer studies are preserved in the format of archived formalin-fixed paraffin-embedded (FFPE) samples. Profiling histone modifications in archived FFPE tissues is critically

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    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: 24, Q&A: 0

    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

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    A Highly Sensitive Method to Efficiently Profile the Histone Modifications of FFPE Samples
    [Abstract]

    The majority of biopsies in both basic research and translational cancer studies are preserved in the format of archived formalin-fixed paraffin-embedded (FFPE) samples. Profiling histone modifications in archived FFPE tissues is critically important to understand gene regulation in human disease. The required input for current genome-wide

    ...
    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: 24, 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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    A Molecular Cloning and Sanger Sequencing-based Protocol for Detecting Site-specific DNA Methylation
    Authors:  Wei Guo, Anthony Cannon and Damon Lisch, date: 05/05/2022, view: 476, Q&A: 0
    [Abstract]

    DNA methylation is a conserved chemical modification, by which methyl groups are added to the cytosine of DNA molecules. Methylation can influence gene expression without changing the sequence of a particular gene. This epigenetic effect is an intriguing phenomenon that has puzzled biologists for years. By probing the temporal and spatial patterns

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    An Improved EMSA-based Method to Prioritize Candidate cis-REs for Further Functional Validation
    Authors:  Ting Wu and Gang Li, date: 04/20/2022, view: 448, Q&A: 0
    [Abstract]

    Cells are the complex product of gene expression programs that involve the coordinated transcription of thousands of genes controlled by cis-regulatory elements (cis-REs). Therefore, identification of cis-REs is the key to decipher the mechanisms underlying the regulation of gene expression. Here, we describe a simple and time-effective protocol

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    circFL-seq, A Full-length circRNA Sequencing Method
    Authors:  Zelin Liu and Ence Yang, date: 04/20/2022, view: 780, 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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    An Optimized Tat/Rev Induced Limiting Dilution Assay for the Characterization of HIV-1 Latent Reservoirs
    [Abstract]

    The administration of antiretroviral therapy (ART) leads to a rapid reduction in plasma viral load in HIV-1 seropositive subjects. However, when ART is suspended, the virus rebounds due to the presence of a latent viral reservoir. Several techniques have been developed to characterize this latent viral reservoir. Of the various assay formats

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    Application of Cadaverine to Inhibit Biotin Biosynthesis in Plants
    Authors:  Nicole M. Gibbs, Shih-Heng Su and Patrick H. Masson, date: 04/20/2022, view: 399, Q&A: 0
    [Abstract]

    Biotin is an essential vitamin in plants. However, characterization of biotin deficiency has been limited by embryo lethality in mutants, which can only be rescued by supplementation of biotin. Here, we describe a protocol to characterize biotin deficiency in Arabidopsis thaliana through application of the polyamine cadaverine. Cadaverine

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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: 706, 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: 680, 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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    Conditional Human BRD4 Knock-In Transgenic Mouse Genotyping and Protein Isoform Detection
    Authors:  Michael Paul Lewis, Shwu-Yuan Wu and Cheng-Ming Chiang, date: 04/05/2022, view: 426, Q&A: 0
    [Abstract]

    Bromodomain-containing protein 4 (BRD4) is an acetyl-lysine reader protein and transcriptional regulator implicated in chromatin dynamics and cancer development. Several BRD4 isoforms have been detected in humans with the long isoform (BRD4-L, aa 1-1,362) playing a tumor-suppressive role and a major short isoform (BRD4-S, aa 1-722) having

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