Lightcycler 480 system

qRT-PCR was performed using a LightCycler 480 system (Roche Diagnostics, Mannheim, Germany) as previously reported^{16 (link)}. The mouse primary microglial cells were pre-treated with memantine (5 µM) for 12 h. Cells were washed and the total RNA was extracted using a High Pure RNA Isolation kit (Roche Diagnostics) according to the manufacturer’s protocol, and was subjected to cDNA synthesis using a Transcriptor First Strand cDNA Synthesis kit (Roche Diagnostics). qRT-PCR was performed with primers (TNF: 5′-CTGTAGCCCACGTCGTAGC-3′, 3′-TTGAGATCCATGCCGTTG-5′; CD45: 5′-TCAGAAAATGCAACAGTGACAA-3′, 3′-CCAACTGACATCTTTCAGGTATGA-5′; IL-1β: 5′-AGTTGACGGACCCCAAAAG-3′, 3′-AGCTGGATGCTCTCATCAGG-5′; IL-6: 5′-GCTACCAAACTGGATATAATCAGGA-3′, 3′-CCAGGTAGCTATGGTACTCCAGAA-5′; IL-10: 5′-CAGAGCCACATGCTCCTAGA-3′, 3′-TGTCCAGCTGGTCCTTTGTT-5′; TGF-β: 5′-TGGAGCAACATGTGGAACTC-3′, 3′-GTCAGCAGCCGGTTACCA-5′; Arginase: 5′-GAATCTGCATGGGCAACC-3′, 3′-GAATCCTGGTACATCTGGGAAC-5′). Actin-β of Universal Probe Library (Roche Diagnostics) was used as a house-keeping control gene. All of these primers were used in our previous report^{16 (link)}. The value of mRNA expression for each sample was automatically calculated as the Ratio by a LightCycler 480 system (Roche Diagnostics, Mannheim, Germany). Ratio = 2^−ΔCp, ΔCp = Cp(target) − Cp(reference).

Residual plasma samples were obtained from individuals with DENV like syndrome or suspected ZIKV virus infection from November 2015 to May 2016 in the states of Tocantins and Alagoas in the North and Northeast regions of Brazil, respectively. To investigate the impact of input sample volume on diagnostic sensitivity, samples were tested for DENV, CHIKV, and ZIKV virus RNA using the research-use Arboplex target-capture transcription-mediated amplification (TC-TMA) assay on the automated Panther system (Grifols Diagnostic Solutions) and the research-use VRI triplex real-time RT-qPCR (rRT-PCR) assay which incorporates the small volume manual RNA extraction method, oligonucleotide sequences and one-step RT-qPCR master mix used for the CDC Trioplex RT-qPCR assay, with amplification performed on a real-time PCR instrument (Roche Lightcycler 480 System). After parallel testing using the Arboplex TC-TMA and VRI triplex RT-qPCR assays, samples with discordant results were re-tested using a primer-probe set and positive control set from the CDC Trioplex RT-qPCR assay (ATCC Item Number NR-50417) on a real-time PCR instrument (Roche Lightcycler 480 System).