Quantifying Interferon-Stimulated Genes in PBMCs

Total RNA from human PBMCs were converted to cDNA (High Capacity RT kit, Life Technologies) and preamplified by limited PCR (PreAmp Master Mix, Life Technologies) with a pool of 96 primers (DeltaGene, Fluidigm). We looked at 38 ISGs in this pool of 96 primers^{53 (link)-56 (link)}. Preamplified cDNAs were treated with ExonucleaseI (New England Biolabs) and diluted to remove unused primers and dNTPs and loaded onto a 96.96 Dynamic Array IFC for real-time PCR analysis on a BioMarkHD (Fluidigm). Analysis was performed, using Fluidigm’s Real Time PCR Analysis Software to determine Ct values, using linear (derivative) baseline correction and auto-detected, assay-specific threshold determination. Ct values were standardized to 3 housekeeping genes (DOCK2, EEF1A1, and FAM105B) that showed high correlation across samples to correct for sample input differences⁵⁷. Four samples from same donor and same draw were run on both Fluidigm assays. The mean of the Ct values were used for analysis. Human sample size was based on availability of IFIH1^NR/NR and IFIH1^R/R subjects.

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Gorman J.A., Hundhausen C., Errett J.S., Stone A.E., Allenspach E.J., Ge Y., Arkatkar T., Clough C., Dai X., Khim S., Pestal K., Liggitt D., Cerosaletti K., Stetson D.B., James R.G., Oukka M., Concannon P., Gale M J.r., Buckner J.H, & Rawlings D.J. (2017). The A946T variant IFIH1 RNA sensor mediates an interferon program that limits viral infection but increases the risk for autoimmunity. Nature immunology, 18(7), 744-752.

Publication 2017

High Capacity RT kit PreAmp Master Mix DeltaGene ExonucleaseI BioMarkHD Real Time PCR Analysis Software

Assays Cdnas Donor Eef1a1 Housekeeping genes Human Primers Real time pcr analysis

Corresponding Organization : University of Washington

Other organizations : University of Florida

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Variable analysis

independent variables

IFIH1 genotype (IFIH1^NR/NR and IFIH1^R/R)

dependent variables

Expression levels of 38 interferon-stimulated genes (ISGs)

control variables

Sample input differences were corrected by standardizing Ct values to 3 housekeeping genes (DOCK2, EEF1A1, and FAM105B)

controls

Positive control: None specified
Negative control: None specified

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