Animals were maintained at 25°C, 75–85% relative humidity and a 18/6 h light/dark cycle.
Growth protocol
Larvae were fed on finely powdered fish food (Tetramind, Tetra Werke, Germany) mixed 1:1 with yeast powder. Adults (males and females) were kept in cages with access ad libitum to raisins and water. For blood feeding, adult female mosquitoes were fed on mice anaesthetized with a mixture of ketamine and xylazine. Males and females were kept together in the cage until the blood meal. Blood-fed females were transferred to a separate cage and kept without males for the remaining duration of the experiment. The transferred females were kept with access to raisins and water and were offered a cup with water as oviposition site at all times. Egg laying was observed between 72 and 96 h PBM. Three samples composed of five mosquitoes each were collected for each experimental time point, the RNA was immediately extracted and stored at -80°C.
Extracted molecule
total RNA
Extraction protocol
Isolated total RNA samples were processed as recommended by Affymetrix, Inc. (Affymetrix GeneChip Expression Analysis Technical Manual, Affymetric, Inc., Santa Clara, CA, USA). In brief, total RNA was initially isolated using TRIzol Reagent (Gibco BRL Life Technologies, Rockville, MD, USA) and purified further by passing it through an RNeasy spin column (Qiagen, Chatsworth, CA, USA). Eluted total RNAs were quantified and a portion of the recovered material adjusted to a final concentration of 1 µg/µl. All starting total RNA samples were quality-assessed prior to beginning target preparation/processing steps by resolving a small amount of each sample (typically 25–250 ng/well) on to a RNA Laboratory-On-A-Chip (Caliper Technologies, Mountain View, CA, USA) that was evaluated on an Agilent Bioanalyser 2100 (Agilent Technologies, Palo Alto, CA, USA).
Label
biotin
Label protocol
Single-stranded, then double-stranded cDNA was synthesized from the poly (A)+ mRNA present in the isolated total RNA (10 µg total RNA starting material each sample reaction) using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA) and poly (T)-nucleotide primers that contained a sequence recognized by T7 RNA polymerase. A portion of the resulting ds-cDNA was used as a template to generate biotin-tagged cRNA from an in vitro transcription reaction, using the BioArray High-Yield RNA Transcript Labeling Kit (T7) (Enzo Diagnostics, Inc., Farmingdale, NY, USA). 15 µg of the resulting biotin-tagged cRNA were fragmented to strands of 35–200 bases in length following prescribed protocols (Affymetrix GeneChip Expression Analysis Technical Manual).
Hybridization protocol
10 µg of this fragmented target cRNA was hybridized at 45 °C with rotation for 16 h (Affymetrix GeneChip Hybridization Oven 640) to probe sets present on an Affymetrix GeneChip® Plasmodium/Anopheles Genome Array. The GeneChip arrays were washed and then stained (SAPE, streptavidin-phycoerythrin) on an Affymetrix Fluidics Station 450.
Scan protocol
Scanning was performed on a GeneChip Scanner 3000. The results were quantified and analysed using GCOS software (version 1.1.1, Affymetrix, Inc.).
Description
A searchable database (Anopheles gambiae Gene Expression Database at UC Irvine) has been made available so that detailed analyses of specific groups of genes based on their descriptions, functions or levels of gene expression variation can be performed. Related publications: Dissanayake, S., Marinotti, O., Ribeiro, J.M.C., & James, A.A. (2006) angaGEDUCI: Anopheles gambiae gene expression database with integrated comparative algorithms for identifying conserved DNA motifs in promoter sequences. BMC Genomics 7:116. & Marinotti, O., Calvo, E., Nguyen, Q. K., Dissanayake, S., Ribeiro, J. M. C., & James, A. A. (2006) Genome-wide analysis of gene expression in adult Anopheles gambiae. Insect Mol Biol 15 (1), 1-12. Marinotti, O., Nguyen, Q. K., Calvo, E., James, A. A., & Ribeiro, J. M. C. (2005) Microarray analysis of genes showing variable expression following a blood meal in Anopheles gambiae. Insect Mol Biol 14 (4), 365-373.
Data processing
The results were quantified and analysed using GCOS software (version 1.1.1, Affymetrix, Inc.) using default values (scaling, target signal intensity = 500; normalization, all probe sets; parameters, all set at default values).
