†These authors contributed equally to this work.
Whole-mount identification of gene transcripts in aphids: Protocols and evaluation of probe accessibility
Article first published online: 14 MAY 2008
Copyright © 2008 Wiley-Liss, Inc.
Archives of Insect Biochemistry and Physiology
Special Issue: Insect Science in Taiwan
Volume 68, Issue 4, pages 186–196, August 2008
How to Cite
Chang, C.-c., Huang, T.-Y., Shih, C.-L., Lin, G.-W., Chang, T.-P., Chiu, H. and Chang, W.-C. (2008), Whole-mount identification of gene transcripts in aphids: Protocols and evaluation of probe accessibility . Arch. Insect Biochem. Physiol., 68: 186–196. doi: 10.1002/arch.20243
- Issue published online: 10 JUL 2008
- Article first published online: 14 MAY 2008
- Manuscript Received:
- Manuscript Accepted:
- National Science Council of Taiwan. Grant Number: 95-2313-B-002-097-MY2
- Program for Academic Comprehensive Promotion of the College of Bio-Resources and Agriculture at the National Taiwan University
- pea aphid;
- whole-mount in situ hybridization;
- Acyrthosiphon pisum
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.