The 8 × 15 K Agilent microarray design chip (A-MEXP-2196) (Mitchell et al., 2012 (link)) was used to detect the set of genes differentially expressed between the resistant population of Vallée du Kou and a susceptible laboratory colony Ngoussou. Each array contains 60 mer probes designed from all 13,000 transcripts of the Ensembl P3.5 A. gambiae genome annotation, plus additional probes for the detoxification genes from a previous microarray design, the ‘detox chip’, used previously to explore metabolic resistance in A. gambiae (David et al., 2005 (link)).
RNA was extracted from three batches of ten females 3 day old A. gambiae s.s. from a F1 sample from the VK6 population (nonexposed to insecticide but known to be resistant to multiple insecticides from bioassays results of VK) and from the Ngoussou strain which is fully susceptible to pyrethroids, DDT, carbamates and organophosphate with 100% mortality observed 24 h after 1 h exposure. RNA was isolated using the Picopure RNA isolation kit (Arcturus). The quantity and quality of extracted RNA were assessed using NanoDrop ND1000 spectrophotometer (Thermo Fisher) and Bioanalyzer (Agilent, Santa Clara, CA, USA) respectively. Complementary RNA (cRNA) of each sample was amplified using the Agilent Quick Amp labeling Kit (two-color) following the manufacturer's protocol. cRNA from the VK6 samples were labeled with cy3 dye while the cRNA from the susceptible strain Ngoussou was labeled with the cy5 dye. cRNA quantity and quality were checked before labeling using the NanoDrop and Bioanalyzer. Labeled cRNAs were hybridized to the arrays for 17 h at 65 °C according to the manufacturer's protocol. Five hybridizations between cRNA from VK and Ngoussou were carried out by swapping the biological replicates (Fig. S6).
Microarray data were analyzed using Genespring GX 11.0 software. In order to identify differentially expressed genes, a cut-off of 2-fold-change and a statistical significance of P < 0.05 and P < 0.01 were applied. The P values were generated from a t-test against zero using the data from the five hybridizations (Fig. S6) after a multiple testing correction using the Benjamin–Hochberg test. Enrichment analysis was carried out using the Blast2Go software (Conesa et al., 2005; Gotz et al., 2008 ) to detect the major Gene Ontology (GO) terms over-represented among the set of probes up or under-transcribed in the VK population in comparison to the entire microarray chip using a Fisher's test for statistical significance. The microarray data from this study were submitted to Array Express, accession number: E-MTAB-1083.
RNA was extracted from three batches of ten females 3 day old A. gambiae s.s. from a F1 sample from the VK6 population (nonexposed to insecticide but known to be resistant to multiple insecticides from bioassays results of VK) and from the Ngoussou strain which is fully susceptible to pyrethroids, DDT, carbamates and organophosphate with 100% mortality observed 24 h after 1 h exposure. RNA was isolated using the Picopure RNA isolation kit (Arcturus). The quantity and quality of extracted RNA were assessed using NanoDrop ND1000 spectrophotometer (Thermo Fisher) and Bioanalyzer (Agilent, Santa Clara, CA, USA) respectively. Complementary RNA (cRNA) of each sample was amplified using the Agilent Quick Amp labeling Kit (two-color) following the manufacturer's protocol. cRNA from the VK6 samples were labeled with cy3 dye while the cRNA from the susceptible strain Ngoussou was labeled with the cy5 dye. cRNA quantity and quality were checked before labeling using the NanoDrop and Bioanalyzer. Labeled cRNAs were hybridized to the arrays for 17 h at 65 °C according to the manufacturer's protocol. Five hybridizations between cRNA from VK and Ngoussou were carried out by swapping the biological replicates (Fig. S6).
Microarray data were analyzed using Genespring GX 11.0 software. In order to identify differentially expressed genes, a cut-off of 2-fold-change and a statistical significance of P < 0.05 and P < 0.01 were applied. The P values were generated from a t-test against zero using the data from the five hybridizations (Fig. S6) after a multiple testing correction using the Benjamin–Hochberg test. Enrichment analysis was carried out using the Blast2Go software (Conesa et al., 2005; Gotz et al., 2008 ) to detect the major Gene Ontology (GO) terms over-represented among the set of probes up or under-transcribed in the VK population in comparison to the entire microarray chip using a Fisher's test for statistical significance. The microarray data from this study were submitted to Array Express, accession number: E-MTAB-1083.
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