Applied Biosystems™ PureQuant™ Th17 Assay

Applied Biosystems™ PureQuant™ Th17 Assay

The Applied Biosystems PureQuant assays are a family of assays that accurately identify and quantify specific immune cell types in a mixed population of cells. These assays are TaqMan qPCR-based and measure genomic DNA methylation at specific sites that serve as a unique identifier. The assays include standards and controls and each 96-well plate can accommodate up to seven test samples. Results are provided as a percentage of target cell type out of total input cells using a free analysis tool.

PureQuant Th17 Assay utilizes a specific demethylated site in the pro-inflammatory cytokine interleukin 17A (IL17). This cytokine is exclusively produced by T helper 17 cells.

Features

• Proven performance―core technology has been used for identification and quantification of immune cell types for over a decade
• Easily standardized―qPCR method enables consistent results across multiple users, instruments, and sites
• Facilitates regulatory filings―established performance specifications following ICHQ2(R1) guidelines
• Scheduling flexibility―assay can be performed using fresh or frozen cells/gDNA

The assays include all reagents, standards, and controls required to run the assay. Specific components include:

• Calibrator: corrects for variation in bisulfite conversion efficiency. Plasmid-based calibrator harbors the genomic target regions in equimolar ratios and includes GAPDH. Subjected to bisulfite conversion followed by copy number estimation via qPCR standard curve. Allows estimation of assay-specific bisulfite efficiency factor: the ratio between TpG/GAPDH copy numbers measured from the same plasmid.
• Reference: checks assay performance. Consists of genomic DNA extracted from human blood of pooled healthy donors. The reference values represent the expected range of the target cell type. For example, CD8+ cells are generally more abundant than Treg or Th17 cells.
• Standards: determine the unknown copy number. Consist of six dilutions spanning 4-log linear dynamic range (30-31250 copies). Absolute copy numbers can reliably be determined using the slope and intercept of the respective standard curves.

Content

• Dynabeads SILANE Genomic DNA Kit, store at 2°C to 8°CC
• 1 x 2 mL PureQuant qPCR Master Mix, store at 2°C to 8°CC
• 4 x ABS
• THFA, store at room temperature
• PureQuant Th17 Module, store at -15 to -25°C (CpG & TpG TaqMan primers and probes, Reference DNA, Lambda DNA, Standard DNA)
• PureQuant Hot Start PCR
• PureQuant Pre-amplification primer