Itaq universal probe kit

Triturated mosquitoes or tissues were centrifuged and 400 μl of supernatant was used to extract viral RNA (QIAamp Viral RNA Mini Kit, Qiagen). DENV or CHIKV genome copies were quantified using a one-step RT-qPCR with iTaq Universal probe kit (BioRad) and primers and probes targeting the envelope (DENV) [37 (link)] and nsP1 (CHIKV) genes [38 (link)]. The 25 μl reaction mix contained 1 μM of forward and reverse primer, 0.125 μM of probe and 4 μl of RNA extract. Thermal profile started at 50°C for 10 min, 95°C for 1 min and 40 cycles of 95°C for 10 sec and 60°C for 15 sec.
An absolute standard curve was generated by amplifying fragments containing the qPCR targets (one fragment for each virus) using forward primers tagged with a T7 promoter; for DENV we used 5'-TAATACGACTCACTATAGGGCAGGATAAGAGGTTCGTCTG-3' and 5'-TTGACTCTTGTTTATCCGCT-3'; for CHIKV we used 5'-TAATACGACTCACTATAGGGTAGAGCGTTGACCCTACTGA-3' and 5'-AAGGATGCCGGTCATTTGAT-3'. The fragments were reverse transcribed using a MegaScript T7 transcription kit (Ambion) and the total amount of RNA was quantified using a Nanodrop (ThermoScientific) to estimate copy number.

To validate the RNA-seq data, expression levels of seven genes (ATH1, DUR1,2, GAP1, MLS1, VMA22, B1J91_K00825 g and B1J91_K02035 g) were measured with qPCR. One µg of RNA extracted from infected bladders or in vitro grown cells was converted into cDNA using the PrimeScript™ RT Reagent Kit (Takara) according to manufacturer’s instructions. qPCRs were carried out using 0.2 µM of each primer and 0.2 µM of the TaqMan probe, using the iTaq Universal Probe kit (Biorad) according to manufacturer’s instructions. Sequences of primers and probes are listed in Table S2. Fold change expression was calculated using the ΔΔCt method, amplifying RDN58 as the housekeeping gene. qPCRs were carried out with a Quantstudio 3 device.