Quantifying Inflammatory Cytokine Expression

Total viral RNA was extracted directly from cell culture supernatants using the QIAamp viral RNA Mini kit (Qiagen) and measurement of HIV transcripts was performed as previously described (82 (link), 89 (link)). Total RNA was isolated from cell pellets using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. RNA concentration was assessed using a NanoDrop 1000. cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions. Quantitative polymerase chain reaction (qPCR) was performed using a Roche LightCycler 480 with the TaqMan Fast Advanced Master Mix and commercial probes for the targets IL1B (Hs01555410_m1; FAM-MGB), IL6 (Hs00174131_m1; FAM-MGB), PTGES (Hs00610420_m1; FAM-MGB), PTGES3 (Hs04187819_g1; FAM-MGB), PTGS1 (Hs00377726_m1; FAM-MGB), PTGS2 (Hs00153133_m1; FAM-MGB), TNF (Hs00174128_m1; FAM-MGB), TREM1 (Hs00218624_m1; FAM-MGB), and the reference gene POLR2A (Hs00172187_m1; VIC-MGB) (all Applied Biosystems). Relative quantification of the target gene expression compared to reference gene expression was performed using the Pfaffl method (90 (link)). Data were then normalised to the vehicle and log₂ transformed.