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Medium-Throughput RNA In Situ Hybridization of Serial Sections from Paraffin-Embedded Tissue Microarrays

  • Edith Francoz
  • Philippe Ranocha
  • Christophe Dunand
  • Vincent BurlatEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1933)

Abstract

(m)RNA spatiotemporal pattern of distribution is of key importance to decipher gene function. In this post-genomic era, numerous transcriptomic studies are made publicly available, sometimes reaching a tissular resolution and even more rarely the cellular level. This “one tissue-numerous genes” information can be completed by the reverse “one gene-numerous tissues” picture through traditional RNA in situ hybridization (ISH). Here, we present a method including (1) principles of transcriptomic data mining to be performed prior and following ISH and (2) a detailed step-by-step medium-throughput ISH protocol performed on serial sections from tissue microarrays. In a recent work, we implemented this method for 39 selected genes studied by medium-throughput ISH complementing an existing tissue-specific transcriptomic dataset focused on the model plant Arabidopsis seed development kinetics (Francoz et al., Scientific Reports 6:24644, 2016). This full integration of ISH and transcriptomics demonstrated the complementarity of both techniques in terms of tissue/cell specificity, signal sensitivity, gene specificity, and spatiotemporal resolution.

Key words

Medium-throughput RNA in situ hybridization Tissue-specific transcriptomics Plants Arabidopsis Seed development Tissue microarray paraffin serial sections Digoxigenin-labeled riboprobes Slide scanner Data integration 

Notes

Acknowledgment

Université Paul Sabatier Toulouse 3, Centre National de la Recherche Scientifique (CNRS) and Laboratoire d’Excellence (LABEX) entitled TULIP (ANR -10-LABX-41) granted this work. Nanozoomer virtual microscopy was performed on the Toulouse Réseau Imagerie (TRI, FR3450). We thank Josiane CHOURRE for help in tissue fixation and infiltration.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Edith Francoz
    • 1
  • Philippe Ranocha
    • 1
  • Christophe Dunand
    • 1
  • Vincent Burlat
    • 1
    Email author
  1. 1.Laboratoire de Recherche en Sciences VégétalesUniversité de Toulouse, CNRS, UPSCastanet TolosanFrance

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