stock: Ama-KTT/M/2 tissue: ten larvae food: containing the LC-50 of α-amanitin for Ama-KTT/M/2 developmental stage: late third instar
Treatment protocol
To identify the genes that are inducible by α-amanitin, one group of Ama-KTT/M/2 larvae were raised on 1.5 mg of α-amanitin per g of food, which is the approximate LC50 concentration of this stock. All other larvae were raised on non-toxic food.
Growth protocol
Freshly-hatched first-instar larvae of the sensitive Canton-S stock and the resistant Ama-KTT/M/2 stock were placed in groups of ten into 2 mL plastic test tubes (USA Scientific, Orlando, FL), containing 500 mg of food. All larvae were raised until they reached the late third instar at 25° C, 70% humidity, and a 12:12 hour light:dark cycle.
Extracted molecule
total RNA
Extraction protocol
Groups of ten late, but still feeding third-instar larvae were randomly picked from across all tubes and flash-frozen in batches of ten in liquid nitrogen, each batch providing the RNA for one microarray chip. All samples were collected on the same morning. RNA extraction was performed without delay, using the RNeasy microarray tissue kit (Qiagen) according to the manufacturer’s instructions.
Label
biotin
Label protocol
Prior to labeling, total RNA samples were checked for purity and concentration, using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher, Waltham, MA, USA) and for integrity, using RNA 6000 Nano Chips in a BioAnalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). cDNA for hybridization was synthesized and biotin labeled from 400 ng of total RNA using a MessageAmp Premier IVT kit (Ambion, Austin, TX, USA) according to the manufacturer's specifications.
Hybridization protocol
Biotinylated cDNA was fragmented, then hybridized, washed, and stained using a GeneChip Hybridization, Wash, and Stain Kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer's specifications.
Scan protocol
Arrays were post-processed on the AFX 450 Fluidics Station before they were scanned on an AFX GC3000 G7 Scanner (Affymetrix, Austin, TX, USA).
Description
Ama-KTT/M/2 is an isochromosome stock for chromosomes 2 and 3 derived from the Ama-KTT stock available from the Drosophila Species Stock Center at the University of California, San Diego as stock #14021 0231.07
Data processing
Data were extracted from the raw images using the Affymetrix Expression Console v. 1.2 software.
