DOI: 10.5061/dryad.stqjq2c40
title: Gene induction by natural toxins in Drosophila melanogaster

description: The transcriptional response of third instar Drosophila larvae to food
 laced with caffeine, pyrethrum oil or neem oil was assessed using Agilent
 4x44K printed microarrays.  Among the most notable responsive
 genes were those belonging to cytochrome P450 and Ecdysone-like kinase
 gene families.
description: <em>Drosophila melanogaster </em>from the genome
 reference line (<em>y; cn bw sp</em> Bloomington ID 2057) were
 raised on agar/semolina food and set to lay on grape juice laying plates
 with live yeast for 6 hours. Embryos were washed of yeast and placed on 2x
 agar following the protocol of Willoughby <em>et al.</em> 2006
 (https://doi.org/10.1016/j.ibmb.2006.09.004).
 The resulting larvae were allowed to develop at 25C under constant light
 for 4.5 days and transferred to 2x agar laced with neme oil (5 mg/mL from
 Neemoil Australia Imports), pyrethrum oil (5mg/mL Botanical Resources
 Australia), caffeine (1.5mg/mL from Sigma) or onto untreated control
 plates for four hours. Larvae were snap frozen in liquid nitrogen, ground
 in a mixer-mill at 25kHz for 5 minutes and RNA extracted with TriSure
 (Bioline cat no. BIO-38032). Purified mRNA re-suspended in water were sent
 to the Australian Genome Research Facility who reverse transcribed into
 cDNA, labelled it with Cy3 or Cy5 and hybridized to Agilient 4x44k gene
 expression microarrays (018972 Oligo microarray version 1, 4x44k,
 G2519; https://doi.org/10.1016/S0076-6879(06)10002-6 ). Two arrays were used (Barcodes: 251897210115 and 251897210116), to provide a degree of technical replication. A pool of random RNA was prepared by mixing RNA from all four chemical stress conditions (Control, Neem, Caffeine and Pyrethrum). This pool was created to ensure that each transcript in each sample would have to compete with a differently dyed RNA transcript from the pool to hybridise with its microarray probe. This competitive hybridisation approach compensates for differing affinities between the probes and their RNA transcripts. slide Dye Gasket 1 Gasket 2 Gasket 3 Gasket 4 1 Cy5 Control Caffeine Pyrethrum Oil Neem Oil 1 Cy3 Pool Pool Pool Pool 2 Cy5 Pool Pool Pool Pool 2 Cy3 Pyrethrum Oil Caffeine Control Neem Oil The Limma library (Smyth 2005 limma: Linear Models for Microarray Data: https://link.springer.com/chapter/10.1007/0-387-29362-0_23 ) was used in an R script to normalize the arrays.
description: The arrays were originally intended to be set up as technical
 replicates with the gasket order mirrored; the pool RNA in all gaskets
 labeled with Cy5 dye and the sample RNA labeled with Cy3. Due to a
 misunderstanding, the arrays were dye swapped and the gasket order
 randomised. This means that the gasket and dye effects for the Control and
 Pyrethrum groups are confounded. Initially the unwanted dye swap was
 thought to be a concern, but examination of the quality data for the
 arrays showed that there was very little dye effect for the non-confounded
 samples Caffeine and Neem. Therefore the unwanted dye swap does not appear
 to adversely affect the dye effect in the dye/array confounded Control or
 Pyrethrum groups either.