Ovaries were dissected in ice-cold PBS and stored at -80C until used.
Growth protocol
Flies were raised on tandard cornmeal-agar medium at 25C. Newly eclosed virgin females were collected and aged for 4 hours prior to dissection of ovaries
Extracted molecule
total RNA
Extraction protocol
RNA was isolated with TRIzol from approximately 150 ovary pairs per replicate, DNase-treated (Qiagen DNaseI) and cleaned up (Qiagen RNeasy) according to the manufacturer instructions.
Label
biotin
Label protocol
Fifty nanograms total RNA was converted to SPIA amplified cDNA using the Ovation RNA Amplification System, v2 (NuGEN Technologies, Cat. #3100) according to the manufacturer’s recommended protocol. The amplified SPIA cDNA product was purified through a QIAGEN QIAquick PCR Purification column (QIAGEN Cat #28104) according to modifications from NuGEN. 3.75ug of this product were fragmented (average fragment size = 85 bases) and biotin labeled using the NuGEN FL-Ovation cDNA Biotin Module, (NuGEN Technologies, Cat. #4200) per the manufacturer’s recommended protocol.
Hybridization protocol
Biotin-labeled cDNA was mixed with Affymetrix hybridization buffer, placed onto Drosophila 2.0 arrays (Cat #900532), and incubated at 45º C for 18 h with 60 rpms rotation in an Affymetrix Model 640 Genechip Hybridization Oven. Following hybridization, the arrays were washed, stained with streptavidin-phycoerythrin (Molecular Probes, Inc. Eugene, OR), signal amplified with antistreptavidin antibody (Vector Laboratories, Inc., Burlingame, CA) , and stained again with streptavidin-phycoerythrin (Molecular Probes, Inc., Eugene, OR), using the Affymetrix Model 450Fluidics Station (Affymetrix, Inc.) following the appropriate fluidics script protocol for the array
Scan protocol
Arrays were scanned with the Affymetrix Model 3000 scanner with 7G upgrade.
Description
Gene expression data from su(Hw)f/v (fertile) young virgin female Drosophila embryos
Data processing
Data were collected using the using the GeneChip operating software (GCOS) v1.4 and expression value estimates were generated using the microarray suite (MAS) v5.0 software.
