Estimation of gene expression of key P450s in triple mutant
haplotype
To determine whether the presence of the triple mutant haplotype type
was associated with differential expression of genes, we examined
individual females from the BusiaUg colony (Triple mutant Freq. 0.297;
95%CI 0.233-0.370). Two legs were removed from individual mosquitoes
for DNA analysis with the remaining mosquito kept for RNA analysis. DNA
was extracted from legs by boiling in STE buffer at 95°C for 90 minutes
and individuals were genotyped using the LNA qPCR assays. RNA was then
extracted individually from 8 mosquitoes in each genotypic group -
homozygotes for the triple mutant haplotype, wild-type homozygotes, and
heterozygotes, using the Arcturus Picopure RNA isolation kit
(Thermofisher). We then performed SYBR green based qPCR to measure the
expression of Cyp6aa1 and Cyp6p4 together with the known
resistance-linked variant Cyp6p3 using the housekeeping genes 40s
ribosomal protein S7 (AGAP010592) and elongation factor Tu (AGAP005128)
for normalisation. The ΔΔCT values were tested for normality and
homogeneity of variances using the Shapiro-Wilks test, and the Bartlett
test, respectively. A significant difference in gene expression between
the genotypic groups was determined by a two-tailed two-sample Student’s
t-test on ΔΔCT values, with a threshold of P=0.05.