Assessing Type I Interferon Induction in Lupus

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.

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Mohan S., Barsalou J., Bradley T.J., Slorach C., Reynolds J.A., Hasni S., Thompson B., Ng L., Levy D., Silverman E, & Kaplan M.J. (2015). Endothelial progenitor cell phenotype and function are impaired in childhood-onset systemic lupus erythematosus. Arthritis & rheumatology (Hoboken, N.J.), 67(8), 2257-2262.

Publication 2015

recombinant IFN-α ABI PRISM 7900HT 2x SYBR Green supermix

Bioassay Cdna Double stranded rna Genes Hela cells Housekeeping gene Hypoxanthine guanine phosphoribosyltransferase Ifn α Myxovirus Primers Prism Protein Protein kinase Real time pcr Sera Sybr green Tetratricopeptide repeats Type i ifn

Corresponding Organization :

Other organizations : University of Michigan–Ann Arbor, Hospital for Sick Children, National Institute of Arthritis and Musculoskeletal and Skin Diseases

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Variable analysis

independent variables

50% SLE sera (by volume)
50% control sera

dependent variables

Fold-induction of 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)
Fold-induction normalized to the housekeeping gene hypoxanthine guanine phosphoribosyltransferase 1 (HPRT1)

control variables

DMEM/10%FBS medium (negative control)
1 KU/ well recombinant IFN-α (positive control)

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