Environmental Engineering Reference
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and a final extension step at 72°C for 6 min. PCR products were gel-purified with the
QIAquick Gel Extraction Kit (Qiagen) before sequencing on an ABI Prism 3130 XL
Genetic Analyzer (Applied Biosystems) using the same primers.
Microarray Screening of Differentially Transcribed Detoxification Genes
The
Aedes detox
chip DNA-microarray, initially developed by Strode et al. [20] and
recently updated with additional genes, was used to monitor changes in the transcrip-
tion of detoxification genes between the Vauclin and the Bora-Bora strains in 4th-stage
larvae and 3 days-old adults. This microarray contains 318 probes representing 290
detoxification genes including all cytochrome P450 P450s, GSTs, CCEs, and addi-
tional enzymes potentially involved in response to oxidative stress from the mosquito
Ae. aegypti
. Each probe, plus six housekeeping genes and 23 artificial control genes
(Universal Lucidea Scorecard, G.E. Health Care, Bucks, UK) were spotted four times
at different positions on each array.
The RNA extractions, cRNA synthesis and labeling reactions were performed
independently for each biological replicate. Total RNA was extracted from batches
of 30 4th-stage larvae or 30 3 days-old adults (15 males and 15 females) using the
PicoPure™ RNA isolation kit (Molecular Devices, Sunnyvale, CA, USA) accord-
ing to manufacturer's instructions. Genomic DNA was removed by digesting total
RNA samples with DNase I by using the RNase-free DNase Set (Qiagen). Total RNA
quantity and quality were assessed by spectrophotometry using a Nanodrop ND1000
(LabTech, France) and by using a Bioanalyzer (Agilent, Santa Clara, CA, USA). Mes-
senger RNAs were amplifi ed using the RiboAmp™ RNA amplifi cation kit (Molecular
Devices) according to manufacturer's instructions. Amplifi ed RNAs were checked for
quantity and quality by spectrophotometry and Bioanalyzer. For each hybridization, 8
μg of amplifi ed RNAs were reverse transcribed into labeled cDNA and hybridized to
the array as previously described by David et al. [19]. For each life-stage, 3 pairwise
comparisons of Vauclin strain versus Bora-Bora strain were performed with different
biological samples. For each biological replicate, two hybridizations were performed
in which the Cy3 and Cy5 labels were swapped between samples for a total of six
hybridizations per comparison in each life-stage.
Spot fi nding, signal quantifi cation and spot superimposition for both dye channels
were performed using Genepix 5.1 software (Axon Instruments, Molecular Devices,
Sunnyvale, CA, USA). For each data set, any spot satisfying one of the following
conditions for any channel was removed from the analysis: (i) intensity values less
than 300 or more than 65,000, (ii) signal to noise ratio less than 3, (iii) less than 60%
of pixel intensity superior to the median of the local background ± 2 SD. Data fi les
were then loaded into Genespring 7.2 (Agilent Technologies, Santa Clara, CA USA)
for normalization and statistic analysis. For each array, the spot replicates of each gene
were merged and expressed as median ratios ± SD. Data from dye swap experiments
were then reversed and ratios were log transformed. Ratio values below 0.01 were
set to 0.01. Data were then normalized using the local intensity-dependent algorithm
Lowess [66] with 20% of data used for smoothing. For each comparison, only genes
detected in at least 50% of all hybridizations were used for further statistical analysis.
Mean transcription ratios were then submitted to a one-sample Student's t-test against
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