Molecular Biology


    Protocols in Current Issue
    In situ Hybridization of miRNAs in Human Embryonic Kidney and Human Pluripotent Stem Cell-derived Kidney Organoids
    Authors:  Filipa M. Lopes, Susan J. Kimber and Ioannis Bantounas, date: 09/05/2021, view: 400, Q&A: 0

    MicroRNAs are small RNAs that negatively regulate gene expression and play an important role in fine-tuning molecular pathways during development. There is increasing interest in studying their function in the kidney, but the majority of studies to date use kidney cell lines and assess the total amounts of miRNAs of interest either by qPCR or by

    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: 2472, 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 ...
    Mapping mRNA-18S rRNA Contacts Within Translation Initation Complex by Means of Reverse Transcriptase Termination Sites and RNAseq
    Authors:  Irene Díaz-López, René Toribio and Iván Ventoso, date: 08/20/2020, view: 1862, Q&A: 0
    [Abstract] The nucleotides involved in RNA-RNA interaction can be tagged by chemical- or UV-induced crosslinking, and further identified by classical or modern high throughput techniques. The contacts of mRNA with 18S rRNA that occur along the mRNA channel of 40S subunit have been mapped by site-specific UV crosslinking followed by reverse transcriptase ...
    Confocal and Super-resolution Imaging of RNA in Live Bacteria Using a Fluorogenic Silicon Rhodamine-binding Aptamer
    Authors:  Regina Wirth, Peng Gao, G. Ulrich Nienhaus, Murat Sunbul and Andres Jäschke, date: 05/05/2020, view: 4454, Q&A: 0
    [Abstract] Genetically encoded light-up RNA aptamers have been shown to be promising tools for the visualization of RNAs in living cells, helping us to advance our understanding of the broad and complex life of RNA. Although a handful of light-up aptamers spanning the visible wavelength region have been developed, none of them have yet been reported to be ...
    Detection of Individual RNA in Fixed Cells and Tissues by Chromogenic ISH
    Authors:  Meng Jiang, Chen Lin and Rongqin Ke, date: 02/05/2020, view: 2398, Q&A: 0
    [Abstract] Visualization of RNA molecules in situ helps to better understand the functions of expressed genes. Currently, most conventional in situ hybridization methods for visualization of individual RNAs are based on fluorescence detection. Herein we present a chromogenic in situ hybridization protocol for visualization of ...
    Labeling and Isolation of Fluorouracil Tagged RNA by Cytosine Deaminase Expression
    Authors:  Harihar Basnet and Joan Massague, date: 11/20/2019, view: 2581, Q&A: 0
    [Abstract] Tissues are comprised of different cell types whose interactions elicit distinct gene expression patterns that regulate tissue formation, regeneration, homeostasis and repair. Analysis of these gene expression patterns require methods that can capture as closely as possible the transcriptomes of cells of interest in their tissue microenvironment. ...
    A Widely Applicable Urea-based Fluorescent/Colorimetric mRNA in situ Hybridization Protocol
    Author:  Chiara Sinigaglia, date: 09/05/2019, view: 3646, Q&A: 0
    [Abstract] In situ hybridization methods are routinely employed to detect nucleic acid sequences, allowing to localize gene expression or to study chromosomal organization in their native context. These methods rely on the pairwise binding of a labeled probe to the target endogenous nucleic acid sequence–the hybridization step, followed by detection ...
    Enzymatic Synthesis and Fractionation of Fluorescent PolyU RNAs
    [Abstract] The physical properties of viral-length polyuridine (PolyU) RNAs, which cannot base-pair and form secondary structures, are compared with those of normal-composition RNAs, composed of comparable numbers of each of A, U, G and C nucleobases. In this protocol, we describe how to synthesize fluorescent polyU RNAs using the enzyme polynucleotide ...
    Modification of 3’ Terminal Ends of DNA and RNA Using DNA Polymerase θ Terminal Transferase Activity
    Authors:  Trung M. Hoang, Tatiana Kent and Richard T. Pomerantz, date: 06/20/2017, view: 7246, Q&A: 0
    [Abstract] DNA polymerase θ (Polθ) is a promiscuous enzyme that is essential for the error-prone DNA double-strand break (DSB) repair pathway called alternative end-joining (alt-EJ). During this form of DSB repair, Polθ performs terminal transferase activity at the 3’ termini of resected DSBs via templated and non-templated nucleotide addition cycles. Since ...
    In vitro Transcription (IVT) and tRNA Binding Assay
    Author:  Sonja MK Schoenfelder, date: 09/20/2014, view: 12436, 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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