Cfx qpcr system

RNA was extracted with RNeasy Plus kit (Qiagen, #74134) according to the manufacturer’s instructions. Reverse transcription of mRNA was performed using SuperScript™ III Reverse Transcriptase (Invitrogen, 18080-085). For real-time PCR analysis, 1 µl of cDNA (25 ng of starting RNA) was amplified per reaction using the iTaq Universal SYBR Green Supermix (Bio-Rad, 172–5124) and the Bio-Rad CFX qPCR system. Primers for real-time qPCR analysis were listed in supplemental table (Supplementary Table 2).

A TaqMan multiplex assay allowing the concomitant quantification of the CCE gene AAEL023844 (present in both duplication haplotypes) and the control gene AAEL007808 from single mosquitoes within the same qPCR was developed. For each gene, primers and probes were designed using Primer3web v 4.1.0 (Rozen & Skaletsky, 2000) with the AaegL5 assembly as reference genome for assessing specificity. For each gene, exonic regions were targeted in order to limit amplification variations associated with natural polymorphism (Table S2). The assay was then tested on all individuals detected as positive by qPCR representing 27 individuals belonging to seven populations from three countries. Each reaction mixture contained 12.5 µl of qPCR probe Master Mix (Bio‐Rad), 2.25 µl of each primer (10 µM), 0.625 µl of each probe (10 µM), 1.25 µl of nuclease free water, and 1 µl of template DNA (0.5 ng/µl). PCR amplifications were performed on a CFX qPCR system (Bio‐Rad) with cycles set as follows: 95°C for 10 min followed by 40 cycles of 95°C for 10 s and 60°C for 45 s followed by FAM and HEX levels reading (see File S1 for a user guideline on this TaqMan assay).

RNA was reverse transcribed using Multiscribe reverse transcriptase (Thermo Fisher #4311235) with random hexamers. cDNA was diluted to 1:100 and quantified using targeted qPCR primers (Supplementary Materials online) and SYBR select (Life Technologies #4472908) on the Biorad CFX qPCR system. A standard curve was used to determine linear range and efficiency of the primers. Gene expression was internally normalized to ACT1 expression and error propagated for replicates (qPCR analysis, Supplementary Materials online). Unless otherwise noted, gene expression was measure in triplicate reactions for each of two biological duplicates. To the best of our ability, we attempted to process each of the ∼100 evolved clones and ancestral strains at the same time to prevent variation within a biological replicate.

RNA was extracted using a standard acid phenol chloroform extraction (Collart, 2001) and DNased with RQ1 DNase (Promega #M6106) according to manufacturer’s instructions. 1ug of RNA was reverse transcribed using Multiscribe reverse transcriptase (Thermo Fisher #4311235) with random hexamers, except for ncFRE6, for which we used a gene specific RT primer due to the need to measure RNA levels strand-specifically. cDNA was measured using targeted qPCR primers (S1 Text) and SYBR select (Life Technologies #4472908) on the Biorad CFX qPCR system.