Sybr green supermix

Total RNA was extracted from CCF with RNeasy kit (Qiagen, Valencia, CA). The RNA was then reverse transcribed to cDNA following vendor’s instructions (Promega, Madison, WI). Real-time PCR was performed to detect and quantify MMP1, MMP2, MMP8 and MMP9 mRNA using the Step One Plus real-time PCR system (Life Technologies, Grand Island, NY, USA). A 20 μl reaction mixture containing 2 μl of cDNA, 2 μl of forward primer (200 nM), 2 μl of reverse primer (200 nM), and 10 μl of 2X SYBR green super mix (Bio-Rad Laboratories, Hercules, CA) was run at a universal cycle (95 °C for 10 min, 40 cycles at 95 °C for 15 s, and 60 °C for 60 s) in accordance with the manufacturer’s instructions as reported earlier.(33 (link)) β-Actin was used as the housekeeping gene. Each PCR reaction was run in triplicate and repeated at least 2 times. The primer sequences used in PCR analysis are listed in Table 1.

This bioassay has been described elsewhere (13 (link)). In brief, HeLa cells were incubated with DMEM/10%FBS medium (negative control), 1 KU/ well recombinant IFN-α (positive control) (Invitrogen, Carlsbad, CA), 50% SLE sera (by volume), or 50% control sera for 6 hours. RNA was extracted using RNeasy (Qiagen, Venlo, Netherlands) and reverse transcribed to cDNA (Invitrogen). Real-time PCR was performed an ABI PRISM 7900HT (Applied Biosystems, Foster City, CA) using 2x SYBR Green supermix (Bio-Rad, Hercules, CA), in triplicate, to quantify five type I IFN-inducible genes (IFIG) - myxovirus resistance-1 (MX1), double-stranded RNA-activated protein kinase (PRKR), IFN-induced protein with tetratricopeptide repeats (IFIT1), IFN-induced protein-44 (IFI44), and IFN-induced protein 44-like (C1orf-29) – and the housekeeping gene hypoxanthine guanine phosphoribosyltransferase 1 (HPRT1). Primers were obtained from Integrated DNA Technologies (Coralville, IA). Results were averaged, normalized to the housekeeping gene, and plotted as fold-induction against 25 age- and gender-matched HC.

The relative changes in mRNA of hCSFs under different conditions were evaluated using quantitative real-time reverse transcription PCR (qRT-PCR) using QuantStudio 6 Flex™ Real-Time PCR System (Applied Biosystems, Carlsbad, CA). Total RNA was extracted from experimental hCSFs using the RNeasy Mini kit (Qiagen, Valencia, CA). 2 μg RNA was used for cDNA conversion using commercial reverse transcription system (Promega, Madison, WI) as reported earlier (Gupta et al., 2018 (link)). The changes in the mRNA expression of GPx4 (Glutathione Peroxidase 4), BAD (Bcl2-associated agonist of cell death and BAX (Bcl2-like protein 4) were studied. A 20 μL qPCR reaction mixture contained 2 μL of cDNA, 2 μL of 200 nM forward primer, 2 μL of 200 nM reverse primer, 10 μL of 2X SYBR green supermix (Bio-Rad Laboratories, Hercules, CA), and 4 μL of RNAse/DNAse free water. The qRT-PCR was run at universal cycle conditions, including initial denaturation at 95 °C for 10 min, and 40 cycles of denaturation at 95 °C for 15 s, annealing, and extension at 60 °C for 60 s. Primer sequences of the genes were mentioned in Table 1. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene. The relative mRNA expression was calculated using the 2^−ΔΔCt method and reported as relative fold change with respect to the corresponding control values.