Whole-mount identification of gene transcripts in aphids: protocols and evaluation of probe accessibility.
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.
Chang
Chun-che
CC
Department of Entomology, Laboratory for Genetics and Development, College of Bio-Resources and Agriculture, National Taiwan University, Taipei, Taiwan. chunche@ntu.edu.tw
Huang
Ting-Yu
TY
Shih
Chun-Liang
CL
Lin
Gee-Way
GW
Chang
Te-Pin
TP
Chiu
Hui
H
Chang
Wen-Chang
WC
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
D015347:RNA Probes; D053487:DEAD-box RNA Helicases