The nucleotide analogue digoxigenin-11-dUTP is widely used as a nonradioactive marker in a broad range of techniques including dot blots, Southern and Northern blots, colony and plaque screenings and in situ hybridizations. In this report, we describe the incorporation of this molecule into a cDNA synthesized by reverse transcriptase as a novel application for digoxigenin. This reaction can be performed as a useful control to check the integrity of a bulk mRNA preparation in the construction of a cDNA library, since the ability of mRNA to direct the synthesis of long molecules of first-strand cDNA is a sign of its integrity.
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Talanta
April 2025
College of Life Sciences, Northwest University, Xi'an, 710069, China. Electronic address:
Straightforward genotyping can provide timely diagnostic information for diseases prevention and treatment. Taking advantages of speediness and convenience, although numerous genotyping strategies combined loop-mediated isothermal amplification (LAMP) and lateral flow have been reported to satisfy the demand of point-of-care test, the false positive result caused by aerosol and primer dimer as an innate conflict seriously limits their practical application. In this study, both aerosol and primer dimer as extrinsic and intrinsic inducements respectively are first broken through at one stroke based on an integrated immunochromatographic biosensor.
View Article and Find Full Text PDFCold Spring Harb Protoc
February 2024
Genome Sciences, University of Washington, Seattle, Washington 98105, USA
Precisely where and when a given gene is expressed is crucial for our understanding of developmental and cell biology but determining this is often constrained by detection limits. Here, we describe a technique for visualization of low-copy mRNA in embryos using tyramide signal amplification (TSA). In this protocol, an anti-sense digoxigenin-labeled RNA probe is hybridized to mRNA in situ.
View Article and Find Full Text PDFDev Growth Differ
January 2023
Laboratory of Animal Regeneration Systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kanagawa, Japan.
Myosin heavy chains (MyHCs), which are encoded by myosin heavy chain (Myh) genes, are the most abundant proteins in myofiber. Among the 11 sarcomeric Myh isoform genes in the mammalian genome, seven are mainly expressed in skeletal muscle. Myh genes/MyHC proteins share a common role as force producing units with highly conserved sequences, but have distinct spatio-temporal expression patterns.
View Article and Find Full Text PDFMethods Mol Biol
June 2022
Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, Toulouse INP, Toulouse, France.
As a mean to cope with their potential cytotoxicity for the host plant, secondary metabolisms are often sequestered within specific cell types. This spatial organization may reach complex sequential multicellular compartmentation. The most complex example so far characterized is the sequential multicellular biosynthesis of the anticancer monoterpene indole alkaloids in Catharanthus roseus.
View Article and Find Full Text PDFSTAR Protoc
March 2022
Centre for Developmental Neurobiology, New Hunt's House, 4.28, King's College London, London SE1 1UL, UK.
This protocol describes a hybridization-proximity labeling (HyPro) approach for identification of proteins and RNAs co-localizing with a transcript of interest in genetically unperturbed cells. It outlines steps required for purification of a recombinant HyPro enzyme, hybridization of fixed and permeabilized cells with digoxigenin-labeled probes, HyPro enzyme binding, proximity biotinylation, and downstream analyses of the biotinylated products. Although the protocol is optimized for relatively abundant noncoding transcripts, recommendations are provided for improving the signal-to-noise ratio in case of scarcer RNA "baits.
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