Quantstudio 5 real time system

Expression levels of EspIgR in the differently treated hemocytes were determined with quantitative RT-PCR (qRT-PCR; primers shown in Table S1) using a QuantStudio 5 Real-Time System (Applied Biosystems, USA) and SYBR Premix Ex Taq (Tli RNaseH Plus; TaKaRa, Osaka, Japan). Reaction conditions were used as following: 94 °C for 3 min, then 40 cycles at 94 °C for 10 s and 60 °C for 1 min, followed by melting from 65 to 95 °C. The gene expression levels were derived by the 2^−ΔΔCT calculation method and normalized to the control group. Three independent experiments were performed and the results are presented as mean ± SD

For validation experiments, we selected the four miRNAs most likely to target the HLA-G gene (NG_029039.1) and messenger RNA (NM_002127.5) based on the sequence alignment analysis (RNAhybrid v.2.2) (Krûger, Rehmsmeier, 2006 (link)). The representative scheme showing the interaction site between HLA-G and the four miRNAs selected for validation was constructed using the ApE v2.0.61 software (https://jorgensen.biology.utah.edu/wayned/ape/). The TaqMan Advanced miRNA cDNA Synthesis Kit (Life Technologies, Foster City, California, USA), TaqMan Advanced miRNA Assay (reference: miR-191-5p; targets: miR-5096, miR-4516, miR-4488, miR-486-5p; Life Technologies), and TaqMan Fast Advanced Master Mix (Life Technologies) were used according to the manufacturer’s instructions to evaluate the miRNA expression. Reverse transcription PCR (RT-PCR) assays were performed in a SimpliAmp Thermal Cycler (Applied Biosystems, Foster City, California, USA) and quantitative PCR (q-PCR) in a QuantStudio 5 Real-Time System (Applied Biosystems) and 7500 Real-Time System (Applied Biosystems) according to the manufacturer’s instructions.

RT-PCR was performed using the ZDC Multiplex Kit from the Institute of Molecular Biology of Paraná with 38 μl of eluate, in accordance with the manufacturer’s recommended protocol (www.ibmp.org.br/en-us/). In this protocol [28 ], a standardized 96-well microplate is subdivided into four isometric subgroups of 24 wells for the respective target arboviruses: DENV-1/4, DENV-2/3, CHIKV, and ZIKV. Each of these four subgroups on the plate is composed of 23 wells to test the samples (38 µl/4 subgroups = 9.5 µl of sample eluate for each subgroup), and one positive control from the kit itself. For the real-time PCR (qPCR) probes, the ZDC kit uses three triplex reactions and two duplex reactions in the same plate: DENV-1 (HEX), DENV-4 (FAM), and internal control (Quasar 670); DENV-3 (HEX), DENV-2 (FAM), and internal control (Quasar 670); CHIKV (FAM) and internal control (HEX); and ZIKV (FAM) and internal control (HEX) [29 (link)].
RT-PCR was amplified using the QuantStudio 5 real-time system (http://thermofisher.com/) at 51 °C for 30 min, followed by 95 °C for 15 min and 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Results were analyzed using the company’s software, and considered positive when the cycle threshold was ≤ 36 for the analyzed virus and ≤ 28 for the internal control.

RNA preparation from blood collected in PAX tubes was performed using PAXgene Blood RNA kit according to the manufacturer’s instructions (QIAGEN). cDNA was subsequently prepared from 500 ng RNA using Quantitect Reverse Transcription Kit (QIAGEN) according to standard protocols.
Gene expression was analyzed by reverse-transcription quantitative real-time PCR (Rt-qPCR) run on a QuantStudio™ 5 real-time system (Thermo Fisher) with TaqMan® reagents. The qPCR cycler was programmed to run an initiation step 50 °C for 2 min and 95 °C for 2 min followed by 40 cycles of denaturation at 95 °C for 1 s and annealing/extension at 60 °C for 20 s. We used TaqMan® fast advanced master mix at a concentration of 1X and 200 ng cDNA for each reaction, together with TaqMan® gene expression assays HS00427620_ml (TATA-box binding protein, TBP), HS00914057_ml (Importin 8, IPO8) and HS01031979_ml (MERTK) at a concentration of 1X (Thermo Fisher). MERTK expression levels were normalized to the two housekeeping genes, TBP and IPO8, and then to a randomly selected control sample to obtain relative gene expression levels.

Two microliters RNA was used to generate cDNA using Transcriptor (Roche), random hexamer primer, and the manufacturer's instructions. cDNA was used either undiluted, 1/20, or 1/100 for quantitative PCR (qPCR) depending on the gene analyzed. Gene-specific qPCR was performed in triplicate using primers near the 5’ end of the gene. qPCRs were assembled in 5 μl reaction mixtures in a 384-well plate using 2X Power SYBR green master mix (Thermo Fisher Scientific) and reactions were run on the QuantStudio5 Real-Time System (Thermo Fisher Scientific). Relative amounts of DNA were calculated using a standard curve generated from 10-fold serial dilutions of purified genomic DNA ranging from 10 ng to 0.001 ng. Relative amounts of S. cerevisiae transcript were normalized to S. pombe tubulin transcripts. All values are expressed relative to that of either wild type +SM or the Spt3/7 deg +SM strain, which were set to 1.0. Each experiment was performed in two biological replicates (cultures collected on separate days) and three technical replicates (independent measurements of the same qPCR sample). Values for technical replicates were averaged which gave a single value for each biological replicate. The final result is a mean of biological replicates.