Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 539845
Title In situ hybridization method for localization of mRNA molecules in medicago tissue sections
Author(s) Kulikova, Olga; Franken, Carolien; Bisseling, Ton
Source In: Methods in Molecular Biology Humana Press Inc. (Methods in Molecular Biology ) - p. 145 - 159.
DOI https://doi.org/10.1007/978-1-4939-8633-0_11
Department(s) EPS
Laboratory of Molecular Biology
Publication type Peer reviewed book chapter
Publication year 2018
Keyword(s) Alkaline phosphatase (AP) - Amplifier - Branched DNA - Preamplifier - RNA in situ probe sets - ViewRNA™ ISH Tissue Assays
Abstract

Here we describe an in situ hybridization (ISH) method using Invitrogen™ ViewRNA™ ISH Tissue Assay (ThermoFisher Scientific) optimized for Medicago root and nodules sections. The method is based on branched (b)DNA signal amplification technology originally developed for use in microplate format and further adapted for detection of (m)RNAs in mammalian tissue sections. Signal amplification is achieved via a series of sequential hybridizations of linking sequences which are anchored to complementary sequences present on specific oligonucleotide probes. The typical (m)RNA probe set contains ~20 synthetic adjacent oligonucleotide pairs. Each probe is composed of a 20bp primary sequence designed to target sequence of interest and a secondary extended sequence serving as a template for hybridization of a preamplifier oligonucleotide. The preamplifier forms a stable hybrid only if it hybridizes to two adjacent probes. By this principle, background is reduced. Other regions on the preamplifier are designed to hybridize to multiple bDNA amplifier molecules that create a branched structure. Finally, alkaline phosphatase (AP)-labeled oligonucleotides, which are complementary to bDNA amplifier sequences, bind to the bDNA molecule by hybridization. By adding Fast Red substrate, red punctuated precipitates are formed that can be detected by light bright and/or fluorescent microscope. ThermoFisher Scientific (https://www.thermofisher.com/nl/en/home.html) designs and synthesizes probe sets for a gene of interest and Invitrogen™ ViewRNA™ ISH Tissue Assay kits include all components required for pretreatment of plant tissues, hybridization and signal amplification.

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