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View additional product information for PureQuant™ Treg Assay - FAQs (A43675)
20 product FAQs found
ROX is the passive reference.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.
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.
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.
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.
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.
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
The PureQuant assays are manufactured at a site with an ISO9001-certified quality management system.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Primers and probe match just one specific region in the entire genome for each assay.
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.
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.