This unit describes methods for non-isotopic RNA in situ hybridization on embryonic mouse sections. These methods can be used to follow the spatiotemporal dynamics of gene expression in an embryonic tissue of interest. They involve the use of labeled (e.g., digoxygenin, FITC) antisense riboprobes that hybridize to a specific mRNA in the target tissue. The probes are detected using an alkaline phosphatase-conjugated antibody recognizing the label and a chromogenic substrate. This method can be used to: (1) assess the expression of a single gene within a tissue, (2) compare the expression profiles of two genes within a tissue, or (3) compare the distribution of a transcript and protein within a tissue. While this approach is not quantitative, it provides a qualitative assessment of the precise cell types where a gene is expressed, which is not easily achievable with other more quantitative methods such as quantitative PCR.
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- * A new technique called irCLIP-RNP, which combines ultraviolet crosslinking with mass spectrometry, helps identify proteins that associate with RNA and RBPs, revealing intricate protein-RNA relationships.
- * The study also introduced a method called Re-CLIP to explore simultaneous RBP co-binding on specific RNAs, enhancing our understanding of dynamic RNA-protein interactions within cells.
Development of a novel polyprobe for simultaneous detection of six viruses infecting stone and pome fruits.
3 Biotech
September 2020
Plant Virus Laboratory, Department of Biosciences, Jamia Millia Islamia (A Central University), Jamia Nagar, New Delhi, 110025 India.
A biotin-labeled, non-isotopic, novel polyprobe was developed for the simultaneous detection of six viruses viz. apple chlorotic leaf spot virus (ACLSV), apple mosaic virus (ApMV), apple stem grooving virus (ASGV), cherry virus A (CVA), prunus necrotic ringspot virus (PNRSV) and plum pox virus (PPV) infecting stone and pome fruit trees through dot-blot hybridization assay. The sensitivity of the polyprobe was checked by serial dilutions of total RNA extracted from the tissues of infected trees.
View Article and Find Full Text PDFThe respiratory syncytial virus polymerase can perform RNA synthesis with modified primers and nucleotide analogs.
Virology
January 2020
Department of Microbiology, Boston University School of Medicine, Boston, MA, USA; National Emerging Infectious Diseases Laboratories, Boston University, Boston, MA, USA. Electronic address:
Respiratory syncytial virus (RSV) is significant for public health, capable of causing respiratory tract disease in infants, the elderly and the immunocompromised. The RSV polymerase is an attractive target for antiviral drug development, but as yet, there is no high throughput assay for analyzing RSV polymerase activity, specifically. In this study, using a primer elongation assay as a basis, we analyzed the tolerance of the RSV polymerase for modifications at the 5' end of the primer, and nucleotide analogs.
View Article and Find Full Text PDFNon-isotopic RNA In Situ Hybridization for Functional Analyses in Medicago truncatula.
Methods Mol Biol
March 2019
Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV), Ciudad Politécnica de la Innovación Edf. 8E, C/ Ingeniero Fausto Elio s/n, Valencia, E-46011, Spain.
Different strategies have been developed and implemented during the last decades aiming to decipher the function of particular genes. Among the different techniques, in situ hybridization of mRNA remains an essential experiment to fully understand gene function. Here, we describe a protocol for the in situ localization of gene transcripts in plants.
View Article and Find Full Text PDFNon-isotopic Method for In Situ LncRNA Visualization and Quantitation.
Methods Mol Biol
October 2016
Affymetrix, 3420 Central Expressway, Santa Clara, CA, 95051, USA.
In mammals and other eukaryotes, most of the genome is transcribed in a developmentally regulated manner to produce large numbers of long noncoding RNAs (lncRNAs). Genome-wide studies have identified thousands of lncRNAs lacking protein-coding capacity. RNA in situ hybridization technique is especially beneficial for the visualization of RNA (mRNA and lncRNA) expression in a heterogeneous population of cells/tissues; however its utility has been hampered by complicated procedures typically developed and optimized for the detection of a specific gene and therefore not amenable to a wide variety of genes and tissues.
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