Gene induction by natural toxins in Drosophila melanogaster

Drosophila melanogaster from the genome reference line (y; cn bw sp 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 et al. 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.