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Applied Biosystems™ PureQuant™ Treg Assay

Catalog No. A43675
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A43675 28 reactions
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Catalog No. A43675 Supplier Applied Biosystems™ Supplier No. A43675

Description

Applied Biosystems™ PureQuant™ assays are a family of assays that accurately identify and quantify specific immune cell types in a mixed population of cells.

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.

The PureQuant™ Treg Assay utilizes a specific demethylated site in the transcription factor FOXP3 gene to positively identify regulatory T-cells. This specific site allows for discrimination of regulatory T-cells from activated CD4+ FOXP3+ conventional T-cells 

Benefits include:

  • 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―regulatory support file provided upon request; established performance specifications following ICHQ2(R1) guidelines
  • Scheduling flexibility―assay can be performed using fresh or frozen cells/gDNA

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

  • Calibrator: 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.

This product is manufactured at a site with an ISO9001-certified quality management system.

Specifications

Quantity 28 reactions
Content And Storage
  • 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 Treg Module, store at -15 to -25°C (FOXP3 & GAPDH TaqMan primers and probes, Reference DNA, Lambda DNA, Standard DNA)
For Use With (Application) Cells, DNA
Shipping Condition Approved for shipment at Room Temperature or on Wet or Dry Ice
Detection Method Primer-Probe Detection

Frequently Asked Questions (FAQs)

What is the passive reference in the PureQuant qPCR Master Mix that is included in the PureQuant Treg Assay (Cat. No. A43675)?

ROX is the passive reference.
In the PureQuant assays, can I make changes to the data analysis template?

No, the template is locked and no changes can be made to the formulas and calculations. In the event of a bad/outlier replicate (or replicates), consult the instruction tab of the template for guidance on performing analysis in these cases.
If I am new to the PureQuant technology, do you have a suitable product that I can use as an orthogonal method for comparison?

Yes, we've developed the corresponding eBioscience Essential Phenotyping kits for flow cytometry characterization of T cells, regulatory T cells, and type 1 and type 17 T helper cells. These kits were used during the development of the PureQuant CD8+, Treg, and Th17 Assays.
Can alternative bisulfite conversion kits or reagents (for example sodium bisulfite) be used for bisulfite conversion?

No, the bisulfite conversation kit was optimized specifically for the PureQuant Assays. The ammonium bisulfite and THFA were selected from specific suppliers and using alternatives will jeopardize performance of the assays.

Do the PureQuant assays require specific qPCR instrumentation to run?

No. The assays have been demonstrated to work on Thermo Fisher qPCR instrumentation and other competitor platforms. The only requirement is that the instrument must be compatible with FAM and non-fluorescent quencher or their respective equivalents.
Since the PureQuant assay technology is licensed-in from a third party, are there any restrictions on its use?

Yes, the Limited Use Label License is as follows:
Notice to Purchaser: The purchase of this product conveys to the purchaser the limited, non-transferable rights under licensed patents for the purchaser to use the product only to (a) perform research and development intended to support the manufacturing of cell therapy products that are intended for subsequent administration to human patients and (b) quality control testing for use in the manufacture of cellular therapy products that are intended for subsequent administration to human patients. This product is not for use in any testing or analysis for any other purpose, any provision of services or any third party commercial use conducting clinical trials monitoring services on patient samples. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. For information on obtaining additional rights, please contact outlicensing@thermofisher.com, Licensing and Commercial Supply, Thermo Fisher Scientific, 5823 Newton Drive, Carlsbad, CA, 92008, United States
What quality management system are the PureQuant assays manufactured under?

The PureQuant assays are manufactured at a site with an ISO9001-certified quality management system.
What are the limits of detection and quantification for PureQuant Methylation assays?

The following summarizes the limit of detection (LoD) and limit of quantification (LoQ) for each PureQuant Methylation assay:

- CD3: LoD 6 and LoQ 17
- CD4: LoD 11 and LoQ 37
- CD8: LoD 19 and LoQ 52
- GAPDH: LoD 7 and LoQ 21
- FOXP3: LoD 3 and LoQ 12
- Th17 CpG: LoD 35 and LoQ 73
- Th17 TpG: LoD 38 and LoQ 73
- B cells: LoD 7 and LoQ 22
- Monocytes: LoD 17 and LoQ 28

Values are represented as copy number. The limits have been determined using TaqMan Universal Master Mix II, no UNG and the QuantStudio 12K Flex Real-Time PCR instrument except for the CD3 Assay. The CD3 Assay utilized TaqMan Environmental Master Mix 2.0.
In the PureQuant Assay, what should I do if the Analysis Template does not work?

Digital security software within many organizations may flag the Analysis Template as “foreign” and hence will preclude it from functioning. Your organization’s IT support may be required in order to make the Template functional.
FOXP3 is on the X chromosome. Why does PureQuant Treg Assay analysis not differentiate between male and female cells?

There are two ways to estimate % Treg: a) Conservative method and b) GAPDH method.

Conservative method: There is only one demethylated copy per FOXP3 positive cell for both male and female donors. FOXP3 is located on the X chromosome and males have one demethylated FOXP3 locus, whereas females have one demethylated plus one methylated FOXP3 gene per cell. Methylated FOXP3 does not contribute to qPCR signal amplification.

GAPDH method: Tregs only have one copy of demethylated (or active) FOXP3 gene regardless of the sex of the donor, but two copies of GAPDH genes, which are demethylated in all leukocytes including Tregs.

Thus, the sex of the donor only matters if Treg is estimated by the conservative method. PureQuant Methylation Treg Assay follows the GAPDH method, hence does not differentiate between male and female cells.
In the PureQuant Assay, what do I do if my controls fail QC, but the other qPCR components pass?

If the controls fail QC, the assay is invalid. One of the primary reasons for failing control QC is improper bisulfite conversion. This could be due to inappropriate volume of ammonium bisulfite/THFA added to the reaction, or inappropriate incubation time/temperature.

In the PureQuant Assay, how do I obtain copy numbers from my qPCR for unknowns?

We recommend referring to your instrument manual for setting up an absolute quantification reaction using a standard curve. Two separate standard curves, one for target cell type and the other for total cell counts, should be used to calculate respective copy numbers for each target.

In the PureQuant Assay, what do I do if my samples fail QC?

Sample QC metrics are solely based on consistency of template loading. High CV will cause the sample to fail. If a sample fails QC due to one “bad” replicate, it is possible to replace the value in the analysis template with the average of two good replicates. If the cell value is simply deleted, the calculation will include the cell as (0) and automatically fail QC. We do not recommend removing/omitting an entire set of replicates.

In the PureQuant Assay, how do I obtain copy number from an adjusted standard curve?

Instrument software can re-analyze copy number from an adjusted standard curve. The analysis template does NOT calculate copy number for samples and controls based on the standard curve. Additional instructions are provided in the first tab of the Analysis Template.
In the PureQuant Assay, what do I do if my standard curve fails QC?

All dilutions of the Standard must be included in the analysis in order to generate a valid result. The analysis template will not work properly if a standard dilution is omitted. It is likely that lower copy number standards will produce a higher standard deviation. In the event of a failing QC, users may need to recalculate the linear regression by omitting bad/outlier replicates for one of the standard dilutions. Unknown copy number must be re-estimated and can be done automatically with the instrument software, or manually. We recommend exercising extreme caution and consistency while pipetting the template and avoiding cross-contamination between wells.
What is the Reference, and why is it included in the PureQuant Assay?

Reference consists of genomic DNA extracted from human blood of pooled healthy donors. The reference values represent the expected range within the target cell type. For example, CD8+ cells are in general more abundant than Treg+ or Th17+ cells. Limits for each assay have been established through rigorous experiments. Experimental values falling outside the specified range make the assay invalid.
What is the Calibrator and why is it included in the PureQuant Assay?

Calibrator is an artificially constructed plasmid that harbors assay target sequences in equimolar ratio. The target sequences are unmethylated and have not been subjected to bisulfite conversion. The Calibrator allows estimation of assay-specific bisulfite efficiency factor (i.e., calibration factor), which is the ratio between target/GAPDH copy numbers measured from the same plasmid. Thus, Calibrator serves as a quality control metric for the PureQuant Assay.

In the PureQuant Assay, how many targets does the PCR primer/probe bind to within the genome?

Primers and probe match just one specific region in the entire genome for each assay.
How does the PureQuant Assay quantify a particular cell type?

The assay utilizes two sets of Standards to generate two standard curves. All the samples and controls must be evaluated in parallel using both the Standards. One standard curve estimates the copy number for the cell type of interest. The other standard curve estimates the total cell number. The cell type of interest is normalized by dividing the target copy number by total cell number.

How does the PureQuant Assay identify a particular cell type?

The assay measures demethylation of genomic DNA at a unique site, which serves as an identifier for that particular cell type. Genomic DNA isolated from cells is subjected to bisulfite conversion followed by setting up of a qPCR standard curve using methylation-specific primers.

For Research Use or Manufacturing of Cell, Gene, or Tissue-Based Products. CAUTION: Not intended for direct administration into humans or animals.