Thu0511 comparison of Paxgene and Tempus Whole Blood Rna Collection and Isolation Systems for the Quantification of Type I Interferon-Stimulated Gene Expression

Abstract

Background:Type I interferons (IFN) have important roles in many pediatric and adult rheumatic diseases and are a new therapeutic target for which several “anti-interferon (anti-IFN)” treatments are currently in use or in development. Since the direct detection of these proteins in biological samples has proved challenging, indirect methods are often used to infer the presence of type I IFN. Most commonly this involves quantification of the relative expression of interferon-stimulated genes (ISGs) that are used to calculate an interferon score (IS) (1). This score has been used for example to asses type I IFN activity in pediatric patients with type I interferonopathies, systemic lupus erythematosus, dermatomyositis and systemic juvenile idiopathic arthritis (2). Both qPCR and Nanostring technology have similar sensitivity and reproducibility for IS determination (3). The use of different whole blood RNA collection systems on the IS have not been evaluated however despite evidence of method-dependent changes in gene expression (4).Objectives:The aim of the study was to compare expression of six common ISGs (IFI27, IFI44L, IFIT1, ISIG15, RSAD2, SIGLEC1) and the corresponding IS in RNA derived from two commonly used whole blood RNA collection systems (PAXgene and Tempus).Methods:Whole blood was collected from ten healthy individuals (median age 25.5 years) in sodium heparin tubes and incubated without or with recombinant human interferon alpha 2b (rhIFNα, 2 IU/ml, 4 hrs, 37C°, 5% CO2). Next, samples were divided between PAXgene (PreAnalytiX, Becton Dickinson) and Tempus (Applied Biosystems) tubes and RNA was isolated according to the manufacturer’s protocols. cDNA was synthesized (∼500ng input RNA; qScript cDNA synthesis kit) and ISG expression measured on a QuantStudio 6 Real-Time PCR instrument using a TaqMan Fast Advanced Assay. For each ISG, expression was normalized against the geometric mean of two housekeeping genes (18s rRNA and HPRT1) and calculated using the formula 2-ΔCt. Relative gene expression is reported as the normalized expression of each ISG divided by the median of normalized expression of the same ISG in unstimulated samples. The median relative expression of all six ISGs was used to calculate the IFN score for each sample.Results:There was no statistically significant difference in the normalized expression of any of the six ISGs in either the rhIFNα-stimulated or unstimulated samples derived from PAXgene or Tempus tubes. The greatest difference in mean normalized expression in both unstimulated and stimulated samples was observed for ISG15 (difference in mean normalized expression was 0.0034 and 0.11, respectively). Overall there was a strong correlation of the IFN score between PAXgene and Tempus tubes for both the unstimulated (R2 = 0.9117, p<0.0001) and and rhIFNα-stimulated samples (R2 = 0.8529, p=0.0001).Conclusion:Despite reported differences in gene expression patterns associated with samples collected in PAXgene versus Tempus tubes, our results demonstrate that 6-gene interferon scores ...

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“…However, there was no significant correlation of oxidative burst activity to the age, disease activity and coexistent infections. (4).…”

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“…Both qPCR and Nanostring technology have similar sensitivity and reproducibility for IS determination (3). The use of different whole blood RNA collection systems on the IS have not been evaluated however despite evidence of method-dependent changes in gene expression (4).…”