Overview
The ESR1 gene encodes estrogen receptor (ER) subtype alpha. The ddPLEX ESR1 Mutation Detection Kit quantitatively interrogates seven key ESR1 variants across three exons: exon 5 (E380Q), exon 7 (S463P) and exon 8 (L536R, Y537C, Y537S, Y537N and D538G), and their wild-type counterparts.
The kit consists of sufficient reagents for 120 reactions (including 60 reactions of each for the Mutant Assay and Wild-Type Assay), a synthetic DNA based ESR1 Positive Control, multiplex supermix, and DTT.
The kit is used with the Bio-Rad QX600™ Droplet Digital™ PCR System or QX600™ AutoDG Droplet Digital™ PCR System and is compatible with QX Manager Standard Edition and Premium Edition Software for data analysis.
Features & Benefits
- Highly sensitive (0.01%–0.025% VAF*) single-well discrimination of seven key ESR1 gene mutations
- Same-day results with a streamlined workflow
- Flexible sample input of DNA from plasma or FFPE
- Kit includes positive and internal controls
- Sensitivity increased with patented wild-type suppression chemistry**
- ddPCR-powered reproducibility and specificity using the QX600 ddPCR system and QX Manager Software
* Variant Allele Frequency
** Wild-type suppression chemistry (i.e., blocker displacement amplification (BDA) technology) is licensed under one or more patents licensed to Bio-Rad by NuProbe USA, Inc.
Performance
Analytical Sensitivity
The limit of blank (LoB) was established for the ddPLEX ESR1 Mutant assay using two reagent lots. Fifty-four replicates of wild-type DNA were tested at an input amount of 40,000 copies. The positive droplet counts were rank ordered for each variant and the 95th percentile was determined to be the LoB. Between the two reagent lots, the higher LoB estimate was used.
To establish the limit of detection (LoD) for the ddPLEX ESR1 mutant assay, a titration of synthetic variant DNA in a background of wild-type cell line DNA was tested at a total input amount of 40,000 copies into the ddPLEX ESR1 mutant assay. Variant allele frequencies were 0.5, 0.2, 0.1, 0.05, 0.025, and 0.01%. Eight replicates of each of the samples for each ESR1 variant were tested. The LoD was determined as the lowest variant allele frequency at which all replicates were above the LoB. A summary of the LoB and LoD results is provided in Table 1.
Table 1. Summary of LoB and LoD results for the ESR1 Mutant Assay
| Target | Limit of Blank (# of Positive Droplets) |
Limit of Detection (Variant Allele Frequency) |
Limit of Detection (Mutant Copies in 40,000 Total) |
|---|---|---|---|
| E380Q | 0 | 0.025% | 10 |
| D538G | 1 | 0.01% | 4 |
| Y537S | 1 | 0.025% | 10 |
| L536R | 1 | 0.025% | 10 |
| Y537N | 0 | 0.025% | 10 |
| Y537C | 1 | 0.025% | 10 |
| S463P | 1 | 0.025% | 10 |
Analytical Specificity
Samples with each of the ESR1 variants at 0.1% in a background of wild-type cell line DNA were tested at an input amount of 40,000 copies into the ddPLEX ESR1 mutant assay. Values below the LoB were considered as negative. The concentration of positive targets was plotted (see Figure 1). Specific detection of the spiked variant is observed with low non-specific detection.
Figure 1. Analytical specificity of the ddPLEX ESR1 mutant assay
Reproducibility
The reproducibility of the ddPLEX ESR1 Kit was assessed by testing samples with 1% and 5% allele frequency at a total input amount of 10,000 copies into the mutant assay and 4,000 copies into the wild-type assay. Samples were prepared with synthetic ESR1 mutant DNA mixed with cell line DNA. Two operators tested each sample in quadruplicate with two different reagent lots across two QX600 instruments. For each sample and target, the mean sample concentration (back calculated from the reported reaction concentration), standard deviation, and coefficient of variation (CV) were calculated over all variables. The CV for wild-type targets was less than 12% and for mutant targets was less than 19% for all samples tested (see Table 2).
Table 2. Reproducibility of the ddPLEX ESR1 Assay
| Variant | Variant Allele Frequency (%) |
Mean Sample Conc. (cp/ul) | Coefficient of Variation (%) | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Variant Allele Frequency (VAF) |
53X-WT | 380-WT | 463-WT | Variant | 53X-WT | 380-WT | 463-WT | Variant | |
| D538G | 1% | 1767.2 | 1769.5 | 1709.1 | 20.5 | 8.1 | 8.2 | 7.8 | 11.8 |
| 5% | 1618.8 | 1635.6 | 1566.6 | 106.9 | 10.7 | 10.3 | 11.3 | 4.1 | |
| E380Q | 1% | 1676.9 | 1697.5 | 1593.7 | 20.5 | 7 | 7.9 | 7.4 | 15.9 |
| 5% | 1604.8 | 1638.3 | 1562.6 | 102 | 4.6 | 4.3 | 4.6 | 7.8 | |
| L536R | 1% | 1741.3 | 1764.1 | 1683 | 20.8 | 8.7 | 8.7 | 8.6 | 14.3 |
| 5% | 1645.1 | 1665.1 | 1584.7 | 107.3 | 7.6 | 7.6 | 7.1 | 5 | |
| S463P | 1% | 1744.9 | 1760.3 | 1693.1 | 20.8 | 8.2 | 8.6 | 8.2 | 18.5 |
| 5% | 1695.7 | 1724.6 | 1639.3 | 104.7 | 9.3 | 10 | 8.9 | 6.2 | |
| Y537C | 1% | 1747.1 | 1763.2 | 1702 | 23 | 8.1 | 7.9 | 8.7 | 13.3 |
| 5% | 1561.8 | 1591.4 | 1514.9 | 108.7 | 9.7 | 9.8 | 8.8 | 4.5 | |
| Y537N | 1% | 1753.7 | 1783.2 | 1692.4 | 21.4 | 7 | 6.5 | 7.1 | 15.1 |
| 5% | 1656.2 | 1677.6 | 1606.6 | 106.1 | 9.9 | 10.9 | 9.5 | 7.7 | |
| Y5375 | 1% | 1758.8 | 1775.1 | 1701 | 21.3 | 7.1 | 7.7 | 6.8 | 8.3 |
| 5% | 1643.9 | 1683.6 | 1595.5 | 108.5 | 8 | 7.4 | 7.4 | 5.5 | |
Applications & Uses
- For the detection, discrimination, and quantification of key ESR1 mutations for breast cancer research, from cfDNA in plasma and/or DNA in formalin-fixed paraffin embedded (FFPE) tissue samples.
Included Components
The ddPLEX ESR1 Mutation Detection Kit (17999501) includes the following components for 120 reactions:
| Material Number | Description | Quantity |
|---|---|---|
| 12019648 | ddPLEX ESR1 Mutation Detection Assay
|
1 |
| 12019606 | ddPLEX ESR1 Positive Control, 100 µl | 1 |
| 12005909 | ddPCR Multiplex Supermix, 1.2 ml | 1 |
| 12012171 | Dithiothreitol (DTT), 2 ml | 1 |
The four components above can also be purchased individually.
Configuration Options (or Instrument Compatibility)
The ddPLEX ESR1 Mutation Detection Kit should be used with the QX600 ddPCR System and either the Manual Droplet Generator or Automated Droplet Generator. Equipment, key consumables, and reagents necessary for the QX600 system are listed below (additional items may be needed, please see the User Guide for more details).
Key Droplet Generation & Reader Equipment, Consumables & Reagents
| Description | Catalog Number |
|---|---|
| QX600 Droplet Reader | 12013328 |
| Automated Droplet Generator | 1864101 |
| QX200 Manual Droplet Generator | 1864002 |
| PTC Tempo Thermal Cycler | 12015392 |
| PCR Plate Heat Seal | 1814040 |
| PX1 PCR Plate Sealer | 1814000 |
| ddPCR Plates 96-well Semi-skirted | 12001925 |
| ddPCR Droplet Reader Oil | 1863004 |
| ddPCR Buffer Control for Probes (2X) | 1863052 |
Reagents & Consumables for Automated Droplet Generation Only
| Description | Catalog Number |
|---|---|
| Automated Droplet Generator Oil for Probes | 1864110 |
| DG32 Automated Droplet Generator Cartridges | 1864108 |
| Pipet Tips for Automated Droplet Generator | 1864120 |
| Pipet Tip Bins for Automated Droplet Generator | 1864125 |
Reagents & Consumables for Manual Droplet Generation Only
| Description | Catalog Number |
|---|---|
| Droplet Generator Oil for Probes | 1863005 |
| Droplet Generator DG8 Cartridge Holder | 1863051 |
| DG8 Cartridges for QX100/QX200 Droplet Generator | 1864008 |
| DG8 Gaskets for QX100/QX200 Droplet Generator | 1863009 |
Related Products
- ddPCR Microsatellite Instability (MSI) Kit
- BRAF V600 ddPCR Multiplex Mutation Screening Kit
- KRAS ddPCR Multiplex Mutation Screening Kits
- NRAS ddPCR Multiplex Mutation Screening Kits
- EGFR Exon19 Deletions ddPCR Multiplex Mutation Kit
Related Category
For Research Use Only. Not for use in diagnostic procedures.
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The ddPLEX ESR1 Mutation Detection Kit detects discriminates and quantifies seven key variants in the ESR1 gene and their wild-type counterparts, which allows for calculation of variant allele frequencies in both plasma and tumor tissue samples.
The ddPLEX ESR1 Mutation Detection Assay discriminates and quantifies seven key variants in the ESR1 gene and their wild-type counterparts, which allows for calculation of variant allele frequencies in both plasma and tumor tissue samples. This assay is a component of the ddPLEX ESR1 Mutation Detection Kit (17009501).
The ddPLEX ESR1 Positive Control is a mixture of synthetic DNA consisting of all ESR1 variants and all wild-type targets covered by the ddPLEX ESR1 Mutation Detection Kit. This assay is a component of the ddPLEX ESR1 Mutation Detection Kit (17009501).
1.2 ml (2 x 0.6 ml), 4x supermix, for use in sample preparation for droplet generation in the QX600/QX200 Droplet Digital PCR Systems
2 ml (2 x 1 ml vials), DTT for use in ddPCR Multiplex Supermix in the QX600/QX200 Droplet Digital PCR Systems
Accessories
96-well, thermal cycler for PCR with cables; for research use only.
Modular thermal cycler platform, includes C1000 Touch Thermal Cycler chassis, 96–deep well reaction module, USB flash drive
Kits & Reagents
Pkg of 30, cartridges for use with the Automated Droplet Generator in the QX600/QX200 AutoDG Droplet Digital PCR System
70 ml (10 x 7 ml), oil for use with droplet generator in the QX600/QX200 Droplet Digital™ PCR Systems
Pkg of 24, cartridges for use with the QX200 Droplet Generator
Pkg of 24, gaskets for use with QX200 Droplet Generator
Systems
Discover how the QX600 Droplet Digital PCR System enables 6-color multiplexing, allowing clear discrimination of multiple targets within samples.
Discover how the QX600 AutoDG Droplet Digital PCR System enables and automates 6-color multiplexing for up to 96-samples per run providing clear discrimination of multiple targets.
Software
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QX600 and QX200 System control and analysis software, with traceability for secure environments
Downloads
QX Manager Software Standard Edition, Version 2.1