Whole-mount identification of gene transcripts in aphids: Protocols and evaluation of probe accessibility

Authors

  • Chun-che Chang,

    Corresponding author
    1. Department of Entomology, Laboratory for Genetics and Development, College of Bio-Resources and Agriculture, National Taiwan University, Taipei, Taiwan
    2. Institute of Biotechnology, College of Bio-Resources and Agriculture, National Taiwan University, Taipei, Taiwan
    • Laboratory for Genetics and Development, Department of Entomology, National Taiwan University, No. 27, Lane 113, Roosevelt Rd., Sec. 4, Taipei 106, Taiwan
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    • †These authors contributed equally to this work.

  • Ting-Yu Huang,

    1. Department of Entomology, Laboratory for Genetics and Development, College of Bio-Resources and Agriculture, National Taiwan University, Taipei, Taiwan
    2. Institute of Biochemical Science, National Taiwan University, Taipei, Taiwan
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    • †These authors contributed equally to this work.

  • Chun-Liang Shih,

    Corresponding author
    1. Department of Entomology, Laboratory for Genetics and Development, College of Bio-Resources and Agriculture, National Taiwan University, Taipei, Taiwan
    • Chun-Liang Shih is now at the Graduate Institute of Basic Medical Sciences, Chang Gung University, Taoyuan 333, Taiwan
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    • †These authors contributed equally to this work.

  • Gee-Way Lin,

    1. Department of Entomology, Laboratory for Genetics and Development, College of Bio-Resources and Agriculture, National Taiwan University, Taipei, Taiwan
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  • Te-Pin Chang,

    1. Department of Entomology, Laboratory for Genetics and Development, College of Bio-Resources and Agriculture, National Taiwan University, Taipei, Taiwan
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  • Hui Chiu,

    1. Department of Entomology, Laboratory for Genetics and Development, College of Bio-Resources and Agriculture, National Taiwan University, Taipei, Taiwan
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  • Wen-Chang Chang

    1. Institute of Biochemical Science, National Taiwan University, Taipei, Taiwan
    2. Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
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Abstract

In situ hybridization has become a powerful tool for detecting the temporal and spatial distribution of gene transcripts in prokaryotes and eukaryotes. We report an efficient protocol for whole-mount identification of the expression of mRNAs in the parthenogenetic pea aphid Acyrthosiphon pisum, an emerging model organism with a growing accumulation of genome sequencing data. In addition to steps common for most animal in situ hybridization protocols, we describe processing methods specific to aphids, the accessibility of antisense riboprobes of different lengths in whole-mounted aphids, and signal intensity versus probe lengths. To find substrate combinations that clearly contrast single and double in situ signals in A. pisum, we tested our protocols using riboprobes constructed from two conserved germline markers, Apvasa and Apnanos, and examined colocalized signals in the germaria and developing oocytes. Finally, we propose conditions for stringent permeabilization that may be applied to tissues deep within the aphid embryo. Arch. Insect Biochem. Physiol. 2008. © 2008 Wiley-Liss, Inc.

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