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

• Published in: Archives of insect biochemistry and physiology Vol. 68; no. 4; pp. 186 - 196
• Main Authors: Chang, Chun-che, Huang, Ting-Yu, Shih, Chun-Liang, Lin, Gee-Way, Chang, Te-Pin, Chiu, Hui, Chang, Wen-Chang
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.08.2008
• Subjects: Acyrthosiphon pisum; Animals; antisense riboprobes; Aphids - genetics; apnanos gene; apvasa gene; DEAD-box RNA Helicases - genetics; Embryo, Nonmammalian - embryology; Female; gene expression; Gene Expression Profiling - methods; Gene Expression Regulation, Developmental; genes; genetic markers; germ cells; germline markers; in situ hybridization; In Situ Hybridization - methods; messenger RNA; mRNA; pea aphid; Reproducibility of Results; RNA probes; RNA Probes - analysis; transcription (genetics); whole-mount in situ hybridization
• ISSN: 0739-4462; 1520-6327
• DOI: 10.1002/arch.20243

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.