title: Whole-mount identification of gene transcripts in aphids: protocols and evaluation of probe accessibility.
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.
authors.affiliation: Department of Entomology, Laboratory for Genetics and Development, College of Bio-Resources and Agriculture, National Taiwan University, Taipei, Taiwan. chunche@ntu.edu.tw
authors.forename: Chun-che
authors.initials: CC
authors.lastname: Chang
authors.forename: Ting-Yu
authors.initials: TY
authors.lastname: Huang
authors.forename: Chun-Liang
authors.initials: CL
authors.lastname: Shih
authors.forename: Gee-Way
authors.initials: GW
authors.lastname: Lin
authors.forename: Te-Pin
authors.initials: TP
authors.lastname: Chang
authors.forename: Hui
authors.initials: H
authors.lastname: Chiu
authors.forename: Wen-Chang
authors.initials: WC
authors.lastname: Chang
mesh_terms: D000818:Animals; D001042:Aphids / Q000235:genetics*; D053487:DEAD-box RNA Helicases / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000196:embryology; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D018507:Gene Expression Regulation, Developmental; D017403:In Situ Hybridization / Q000379:methods*; D015347:RNA Probes / Q000032:analysis; D015203:Reproducibility of Results
chemical_list: D015347:RNA Probes; D053487:DEAD-box RNA Helicases