Microarray analysis of juvenile hormone response in Drosophila melanogaster S2 cells
1USDA/ARS Vegetable Crops Research Unit, University of Wisconsin – Madison, Madison WI 53706
2Department of Entomology, University of Wisconsin – Madison, Madison WI 53706
3Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121
4 Current address: INVITROGEN Corporation, 501 Charmany Drive, Madison, WI 53719
Abstract
A microchip array encompassing probes for 14,010 genes of Drosophila melanogaster was used to analyze the effect of juvenile hormone (JH) on genome-wide gene expression. JH is a member of a group of insect hormones involved in regulating larval development and adult reproductive processes. Total RNA was isolated from Drosophila S2 cells after 4 hours treatment with 250 ng/ml (10R) JH III or 250 ng/ml methyl linoleate. A collection of 32 known or putative genes demonstrated a significant change with JH III treatment (r > 2.0, P ≤ 0.005). Of these, the abundance of 13 transcripts was significantly increased and 19 decreased. The expression of a subset of these loci was analyzed by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Three loci that exhibited constant expression in the presence and absence of JH III (RP49 [FBgn0002626], FBgn0023529, and FBgn0034354) were evaluated and found to be reliable invariant reference transcripts for real-time RT-qPCR analysis using BestKeeper and geNorm software. Increased expression in presence of JH III was confirmed by real-time RT-qPCR analysis. However, only one of five loci that exhibited reduced expression on microarrays could be confirmed as significantly reduced (P ≤ 0.05). Among the confirmed JH III up-regulated genes were two loci of unknown function (FBgn0040887 and FBgn0037057) and Epac, an exchange protein directly activated by cyclic AMP, a guanine nucleotide exchange factor for Rap1 small GTPase.
Keywords: real-time RT-qPCR, Epac
Abbreviations: JH, juvenile hormone; Epac, exchange protein directly activated by cyclic AMP; MLA, methyl linoleate; ORF, open reading frame; RT–qPCR, quantitative reverse transcription polymerase chain reaction; RER, relative expression ratio
Correspondence: a dkwillis@wisc.edu,
b jun.wang01@invitrogen.com,
c stanford@entomology.wisc.edu,
d aorth@gnf.org,
e goodman@entomology.wisc.edu
Associate Editor: Zhijian Jake Tu was editor of this paper
Received: 3 January 2008 | Accepted: 11 October 2008 | Published: 15 June 2010
Copyright: This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unrestricted use, provided that the paper is properly attributed.
ISSN: 1536-2442 | Volume 10, Number 66
Willis DK, Wang J, Lindholm JR, Orth A, Goodman WG. 2010. Microarray analysis of juvenile hormone response in Drosophila melanogaster S2 cells. Journal of Insect Science 10:66, available online: insectscience.org/10.66
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