ddPLEX ESR1 Mutation Detection Kit

ddPLEX ESR1 Mutation Detection Kit

The ddPLEX ESR1 Mutation Detection Kit detects 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.

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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

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

Chart about 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 (17009501) includes the following components for 120 reactions and can accommodate up to 42 samples and controls:

Material Number Description Quantity
12019648 ddPLEX ESR1 Mutation Detection Assay
  • ddPLEX ESR1 Mutant Assay (85 µl, 60 reactions)
  • ddPLEX ESR1 Wild-Type Assay (85 µl, 60 reactions)
  • ddPLEX Enhancer (100 µl)
  • ddPLEX Internal Control (500 µl)
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

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For Research Use Only. Not for use in diagnostic procedures.

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ddPLEX ESR1 Mutation Detection Kit
17009501

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.

Image
ddPLEX ESR1 Mutation Detection Assay
12019648

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).

Image
ddPLEX ESR1 Positive Control
12019606

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).

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12005909_thumb.jpg
12005909

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

Accessories

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PX1-PCR-Plate-Sealer-SKU-181-4000-thumb.jpg
(Discontinued) PX1 PCR Plate Sealer
1814000
PCR plate sealer, includes heat sealing instrument, plate support block that holds 96-well and 384-well plates, sealing frame, power cord
Image
185-1196_tn.jpg
1851197

Modular thermal cycler platform, includes C1000 Touch Thermal Cycler chassis, 96–deep well reaction module, USB flash drive

Kits & Reagents

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automated-droplet-generation-oil-for-probes-186-4110-thumb.jpg
1864110
140 ml, oil for use with the Automated Droplet Generator for probe-based detection with the QX200 AutoDG Droplet Digital PCR System
Image
dg32-automated-droplet-generator-cartridges-186-4008-thumb.jpg
1864108

Pkg of 30, cartridges for use with the Automated Droplet Generator in the QX600/QX200 AutoDG Droplet Digital PCR System

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pipet-tips-for-the-autodg-system-186-4120-thumb.jpg
1864120
Pkg of 20, boxes of 96 each, pipet tips for use with the Automated Droplet Generator in the QX200™ AutoDG™ Droplet Digital™ PCR System
Image
pipet-waste-bins-for-the-auto-dg-system-186-4125-thumb.jpg
1864125
Pkg of 10, pipet tip waste bins for use with the Automated Droplet Generator in the QX200™ AutoDG™ Droplet Digital™ PCR System

Systems

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17007769-sku_tn
17007769

Discover how the QX600 Droplet Digital PCR System enables 6-color multiplexing, allowing clear discrimination of multiple targets within samples. 

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17008371
17008371

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. 

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Software

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Downloads

QX Manager Software Standard Edition, Version 2.1
QX-MANAGER-V.2.1-STD

QX Manager Software Standard Edition, Version 2.1

Documents

Number Description Options
10000171375
ddPLEX ESR1 Mutation Detection Kit User Guide
3652
ddPLEX ESR1 Mutation Detection Kit Flier
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