Ifi16

Cellular RNA was isolated using the RNeasy Plus Mini Kit (Qiagen). The RNA was eluted in RNase free water, and the concentration was determined with a Nanodrop 2000 (Thermo Scientific). The qRT-PCR reactions were performed using the TaqMan RNA-to-Ct Master Mix and the Viia7. Reactions (50 µl) were performed in the presence of the master mix, and the following predesigned Human TaqMan assays: APOBEC3A_B (Hs00377444_m1), APOBEC3D (Hs00537163_m1), APOBEC3F (Hs00736570_m1), APOBEC3G (Hs00222415_m1), TRIM5 (Hs01552558_m1), TRIM22 (Hs00232319_m1), SAMDH1 (Hs00210019), and IFI16 (Hs00194261) (Thermo Scientific). Cycling parameters were 48 °C for 20 min, 95 °C for 10 min, then, 45 cycles at 95 °C for 15 s, and 59 °C for 1 min. First, the triplicate samples were pooled together for screening of changes in gene expression of the above genes using 50 ng of RNA in each qRT-PCR reaction. The pooled samples that demonstrated a change in gene expression were further evaluated and each individual sample was examined for gene expression using 25 ng of RNA in each qRT-PCR reaction. DCt values were calculated to normalize the Ct value of the gene of interest as a function of the Ct value of the GAPDH RNA signal. Then fold-change in gene expression was determined by calculating the 2^(-DDCt) for the various conditions in relation to the control (no adjuvant).

Total RNA was isolated using the RNeasy Mini kit following the manufacturer’s instructions (Qiagen, Hilden, Germany). RNA quality and quantification were determined by TapeStation 4200 (Agilent, St. Clara, USA) following the manufacturer’s instructions. Neutrophil total RNA was isolated using RNeasy Mini kit following the manufacturer’s instructions (Qiagen), and complementary DNA (cDNA) was synthesized using M-MLV Reverse Transcriptase (Thermo Fisher Scientific). RT-PCR was performed in duplicates using the cDNA and Platinum Taq polymerase (Thermo Fisher Scientific), 200 nM dNTP (Promega, Southampton, UK), 50mM MgCl₂ (Thermo Fisher Scientific), and TaqMan primer/probe sets (Thermo Fisher Scientific). Samples were matched to a standard curve generated by amplifying serially diluted products using the same PCR reaction and normalized to GAPDH (Hs00266705_g1) to obtain the relative expression value. Real-time assays were run on FX96 Cycler (Bio-Rad). The following are the primes used: STAT1 (Hs01013996_m1), STAT2 (Hs01013115_g1), STAT3 (Hs00374280_m1), IFIH1 (Hs00223420_m1), IFIT (Hs00356631_g1), ISG15 (Hs00192713_m1), MX1 (Hs00895608_m1), IRF1 (Hs00971965_m1), SOCS1 (Hs00705164_s1), USP18 (Hs00276441_m1), IFI44 (Hs00197427_m1), IFI16 (Hs00986757_m1), and OAS (Hs00242943_m1) (all from Thermo Fisher Scientific).