Quantitative Analysis of Viral and Inflammatory Markers in Tissue Samples

Total RNA was extracted from tissues using Trizol (Ambion, 15596018). Tissues were homogenized into Trizol reagent using the FastPrep-24 5 G homogenizer (MP Biomedicals). Total RNA was isolated using the PureLink RNA mini-kit (Ambion, 12183025). Purified RNA was quantified, checked for quality assurance, and then reverse transcribed with the qScript Reverse Transcription kit (QuantaBio, 95047). For quantification of vRNA, a standard curve was generated using 10-fold serial dilutions of ZIKV RNA standard. qRT-PCR for ZIKV prM-E was performed with TaqMan Gene Expression Master Mix, ZIKV Primers, and probe as previously described.^{35 (link)} The standard curve had an R value greater than 0.99. vRNA copies were interpolated from the standard curve using the average Ct value obtained from samples run in triplicate. qRT-PCR for GAPDH, IL-1β, TNF-α, IL-10, SOCS3, MMP2, MMP9, and COX2 were performed with PerfeCTa SYBR Green SuperMix Low Rox (Quantabio, #95056-500). Primers for GAPDH, IL-1β, TNF-α, IL-10, SOCS3, and COX2 were as previously described.^{36 (link)} MMP2 primers were as follows; forward 5ʹ-AACGGTCGGGAATACAGCAG-3ʹ; reverse 5ʹ-GTAAACAAGGCTTCATGGGGG-3ʹ. MMP9 primers were as follows; forward 5ʹ-AACCTCCAACCTCACGGACA-3ʹ; reverse 5ʹ- AGGTTTGGAATCGACCCACG-3ʹ. Data was analyzed using the ∆∆Ct method to determine normalized relative expression.