In situ hybridization method for localization of mRNA molecules in medicago tissue sections

Olga Kulikova, Carolien Franken, Ton Bisseling*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

5 Citations (Scopus)

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.

Original languageEnglish
Title of host publicationFunctional Genomics in Medicago truncatula
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages145-159
ISBN (Electronic)9781493986330
ISBN (Print)9781493986323
DOIs
Publication statusPublished - 1 Jan 2018

Publication series

NameMethods in Molecular Biology
Volume1822
ISSN (Print)1064-3745

Keywords

  • Alkaline phosphatase (AP)
  • Amplifier
  • Branched DNA
  • Preamplifier
  • RNA in situ probe sets
  • ViewRNA™ ISH Tissue Assays

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    Kulikova, O., Franken, C., & Bisseling, T. (2018). In situ hybridization method for localization of mRNA molecules in medicago tissue sections. In Functional Genomics in Medicago truncatula: Methods and Protocols (pp. 145-159). (Methods in Molecular Biology; Vol. 1822). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-8633-0_11