Single-Cell Analysis

Profiling EGFR DNA Mutation Analysis in Circulating Rare Cells
Using the Genesis Cell Isolation System with Celselect Slides™
and Droplet Digital™ PCR (ddPCR™)
ProtocolAbstract

The epidermal growth factor (EGFR) mutation is critical for determining the most effective treatment
for patients with lung cancer. Identifying the mutation in rare circulating tumor cells (CTCs) found inperipheral blood can offer crucial insights into the efficacy of certain drugs and patient prognosis,
especially in non–small cell lung cancer (NSCLC). Unfortunately, the rarity of these cells presents asignificant detection challenge.
The Genesis Cell Isolation System with Celselect Slides can efficiently capture CTCs based on sizeand deformability. These cells can then be recovered for further analysis. To detect these cells,Droplet Digital PCR can be used for direct quantification of DNA mutation.
This optimized protocol using a model CTC sample is designed as a starting point for specific cancerassociated genetic mutation detection. We recommend end users choose and validate their assays
with this protocol before use.
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Workflow Overview
Part 1. Rare Cell Enrichment

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Part 2. EGFR Mutation Detection

Prepare model sample(blood + lung cancer cells)

Assemble PCR reactions
and make droplets

Isolate rare cells

Run PCR

Enrich rare cells

Read droplets

Lyse cells and extract DNA

Analyze data

Profiling EGFR DNA Mutation Analysis in Circulating Rare Cells Using the Genesis Cell Isolation System with Celselect Slides and Droplet Digital PCR (ddPCR)

Instruments and Materials

3.

Instruments*

Mix well with 4 ml of 1x phosphate buffered saline (PBS). Store
this PBS + blood solution at room temperature on a bloodrocker/shaker that is gently agitating until use. If it is longer
than 1 hr, store at 4°C.

■

Genesis Cell Isolation System (catalog #CEL20101)■

QX200™ Droplet Generator (#1864002)■

QX200 Droplet Reader (#1864003)

Celselect Slides Enrichment and DNA Extraction■

QX200 AutoDG™ Droplet Digital PCR System (#1864100) (optional)

1.

■

PX1 PCR Plate Sealer (#1814000)■■

C1000 Touch Thermal Cycler with 96–Deep Well Reaction
Module (#1851197)

Refer to the following sections in the Celselect Slides
Enrichment User Manual (bulletin 3337) to perform CelselectSlides enrichment:
a. Performing the Genesis System Self-Test
b. Celselect Slides and Slide Station Set-Up

Qubit 4 Fluorometer (Thermo Fisher Scientific Inc., #Q33226)

c. Priming the Celselect Slides

Materials*■

Celselect Slides Enrichment Kit (#CEL80110)

d. Preparing the Reagents Cartridge■

DNAzol BD (Molecular Research Center, Inc., #DN129)

e. Preparing the Blood Sample■

ddPCR EGFR Exon 19 Deletions Screening Kit (#12002392)■■

1) Use the blood sample prepared according to instructionsin the Model CTC Sample Preparation section

ddPCR Mutation Detection Assay: EGFR T790M (Assay ID#dHsaMDV2010019)

f. Running the Genesis System1) At step 15, the recovered cell sample can becentrifuged at 600 rcf for 5 min before cell lysis

ddPCR Mutation Detection Assay: EGFR L858R (Assay ID#dHsaMDV2010021)■

Qubit 1X dsDNA High Sensitivity Assay Kit (Invitrogen, #Q33230)■

DG8 Cartridges for QX200 Droplet Generator (#1864008)■

Droplet Generation Oil for Probes (#1863005)■

Automated Droplet Generation Oil for Probes (#1864110) (optional)■

ddPCR Supermix for Probes (No dUTP) (#1863023)■

PCR Plate Heat Seal (pierceable foil seal; #1814040)■

DNA LoBind Microcentrifuge Tube, 1.5 ml (Eppendorf, #022431021)■

ddPCR 96-Well Plates (#12001925)* Source is Bio-Rad Laboratories, Inc. unless otherwise noted.

Rare Cell Enrichment Workflow Using the Genesis System with
Celselect Slides
For EGFR mutation detection using the Genesis System with
Celselect Slides and Droplet Digital PCR, three different model
CTC samples were prepared with three different non–small celllung cancer cell lines at different EGFR mutations.
■■■

NCI-H1975 is a human non–small cell lung cancer cell line
harboring the EGFR L858R mutation and the T790M mutation

2.

3.

Transfer the entire sample to an Eppendorf DNA LoBind 1.5 ml
Microcentrifuge Tube.

4.

Shake vigorously by hand for 15–20 seconds and store atroom temperature for 5 min.

5.

Add 400 µl of isopropanol to the DNAzol BD-lysate
sample mixture.

6.

Vortex or shake vigorously and store at room temperaturefor 4 min.

7.

Sediment the precipitated DNA by centrifugation at 10,000 rcffor 6 min.

8.

Upon centrifugation, remove the supernatant and gently add
0.5 ml of DNAzol BD to the DNA pellet.

9.

Vortex/shake the DNA pellet until completely dispersed.

11. Remove the supernatant and wash the DNA pellet by mixing
with 1 ml of 75% ethanol.

A549 is a human non–small cell lung cancer cell line with thewild-type EGFR

12. Repeat steps 10–11 for a total of two DNA washes.
13. Remove any residual ethanol with a micropipet. Do not allow
the pellet to completely dry.

Prepare a cell stock suspension at a concentration of 100,400, and 1,000 cells/10 µl.

14. Solubilize DNA by adding 20 µl of DNase-/RNase-freedistilled water.

Spike the 10 µl of cell stock solution into 4 ml of whole blood
to prepare a model CTC sample.

© 2023 Bio-Rad Laboratories, Inc.

Remove the supernatant and add 1 ml of DNAzol BD directly
into the conical tube.

10. Centrifuge the resulting mixture at 10,000 rcf for 5 min.

NCI-H1650 is a human non–small cell lung cancer cell line witha deletion mutation on exon 19 of the EGFR geneModel CTC Sample Preparation

1.

2.

15. Measure a dsDNA quantity in the sample by following theQubit 1X dsDNA High Sensitivity Assay Kit protocol.

2Bulletin 3555

Profiling EGFR DNA Mutation Analysis in Circulating Rare Cells Using the Genesis Cell Isolation System with Celselect Slides and Droplet Digital PCR (ddPCR)

EGFR Mutation Detection Workflow Using Droplet Digital PCR
ddPCR Mutation Detection Assays are expertly designed
specifically for Droplet Digital PCR using proprietary computationalalgorithms. The assays used in this protocol consist of mutant
target and wild-type assays mixed and provided in a single
tube, with FAM targeting the mutant allele and HEX targeting thewild-type allele. This assay is made up of a single primer set plus
two competitive probes. This assay takes advantage of the highsensitivity and precision of Droplet Digital PCR.

2.

Proceed to thermal cycling (see Table 2) and subsequentreading of droplets in the QX200 Droplet Reader.

Table 2. Recommended thermal cycling conditions for the C1000 Touch
Thermal Cycler with 96–Deep Well Reaction Module.*Cycling Step

Temperature, °CTimeRamp Rate,°C/sec

Numberof Cycles

Enzyme activation95

10 min21

Denaturation

94

30 sec240

Annealing/extension

55

1 min

240

Enzyme deactivation98

10 min21Reaction Setup

Hold (optional)

4Infinite

11

1.

Thaw all components to room temperature. Mix thoroughly
by vortexing each tube to ensure homogeneity because
a concentration gradient may form during –20°C storage.
Centrifuge briefly to collect contents at the bottom of eachtube and store protected from light.

* For the C1000 Touch Thermal Cycler with 96–Deep Well Reaction Module, use aheated lid set to 105°C and set the sample volume to 40 µl.

1.

After thermal cycling, place the sealed 96-well plate in theQX200 Droplet Reader.

Prepare samples at the desired concentration before setting
up the reaction mix according to the guidelines in Table 1. If
multiple samples are to be assayed using the same target andreference duplex assay, prepare a master reaction mix withoutsample template, dispense equal aliquots into the reaction
tubes, and add the sample template to each reaction tube asthe final step.

2.

Open QX Manager Software Standard Edition to set up a newplate layout according to the experimental design. Refer to theQX200 Droplet Reader and QX Manager Software Standard
Edition User Guide (10000107223).

3.

To add a plate in the QX Manager Software, click the Add Platetab. The software should enable the Configure Plate button.
Click Configure Plate to open the plate configuration window.

4.

Plate features show in the plate configuration window. Fill inthe plate and well information, select the wells, designate thesample name, experiment type, ddPCR Supermix for Probes(No dUTP) as the supermix type, target name(s), target type(s),and reference: Ch1 for FAM and Ch2 for HEX.

5.

Select Apply to load the wells and, when finished, select OK.

6.

Once the plate layout is complete, select Run to begin thedroplet reading process. Select the appropriate dye set andrun options when prompted.

7.

After data acquisition, select samples in the well selector
under Analyze. Set appropriate threshold for the ddPCR
Mutation Detection Assay. For more detailed information about
setting thresholds, please refer to the Rare Mutation Detection
Best Practices Guide (bulletin 6628).

8.

The concentration reported is copies/μl of the final 1x ddPCRreaction.

2.

3.

Data Acquisition and Analysis

Prepare a negative control: at least one well containing
only wild-type template at a concentration similar to theconcentration of unknown samples.

Table 1. ddPCR reaction setup.
Component

Volume per Reaction, μl

Final Concentration

2x ddPCR Supermix forProbes (No dUTP)

101x

20x target (FAM) and wildtype (HEX) primers/probe

11x

Restriction enzyme, diluted

12–5 U/reaction

Variable

Variable

20*–Target sample and/or
DNase-free waterTotal volume* For the Automated Droplet Generator, prepare 22 µl per reaction.

4.

5.

Mix thoroughly by vortexing the reaction tubes. Centrifuge
briefly to ensure that all components are at the bottom of eachreaction tube. Allow the reaction tubes to equilibrate at room
temperature for about 3 min.
Once the reaction mixtures are ready, load 20 μl of eachreaction mix into a sample well of a DG8 Cartridge (#1864008)
followed by 70 μl of Droplet Generation Oil for Probes(#1863005) into the oil wells, according to the QX200 Droplet
Generator Instruction Manual (10031907). For the AutomatedDroplet Generator, follow instructions in the AutomatedDroplet Generator Instruction Manual (10043138).

Results
ddPCR plots of cancer cell lines H1975 (L858R/T790M), H1650(exon 19 deletion), and A549 (wild type) for the three tested EGFR
mutations are shown in Figure 1. For the exon 19 deletion, theBio-Rad ddPCR Mutation Detection Assay screens for 15 common
deletions (nondiscriminatory detection in the FAM channel) but inthis protocol, one of the exon 19 deletions is being tested in theH1650 cell line.

Thermal Cycling Conditions

1.

After droplet generation with the QX200 Droplet Generator,
carefully transfer droplets into a clean ddPCR 96-Well Plateor remove the plate containing ddPCR droplets from theAutomated Droplet Generator. Seal the plate with the PX1PCR Plate Sealer.

© 2023 Bio-Rad Laboratories, Inc.

3

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Profiling EGFR DNA Mutation Analysis in Circulating Rare Cells Using the Genesis Cell Isolation System with Celselect Slides and Droplet Digital PCR (ddPCR)

EGFR T790M
5,000

A549: 100 cells

4,000
FAM amplitude

FAM amplitude

4,000
3,000

Wild type

2,000

5,000A549: 400 cells

1,000

3,000

Wild type

2,000
1,000

0
1,000

2,000

3,000

4,000

5,000

3,000

Wild type2,000
1,00000

A549: 1,000 cells

4,000
FAM amplitude

5,000

00

1,000

HEX amplitude

2,000

3,000

4,000

5,000

6,000

7,000

0

1,000

2,000

HEX amplitude

3,000

4,000

5,000

HEX amplitude

EGFR L858R
7,000

H1975: 100 cells
FAM amplitude

FAM amplitude

5,000

Mutant

4,000
3,000

8,000

H1975: 400 cells

6,000

Wild type

2,000

4,000

Wild type

3,000
2,000
1,000

5,000
4,000

1,000
00–1,000–1,000

2,000

3,000

4,000

5,000

6,000

Wild type

2,000

0

1,000

Mutant

3,000

1,000

0

H1975: 1,000 cells

7,000
6,000

Mutant5,000

FAM amplitude

7,000
6,000

0

1,000

HEX amplitude

2,000

3,000

4,000

5,000

0

1,000

2,000

HEX amplitude

3,000

4,000

5,000

6,000

HEX amplitude

Exon 19 Deletion
8,000

H1650: 100 cells
Mutant6,000

Wild type

5,000
4,000
3,000

FAM amplitude

FAM amplitude

7,000

5,000

1,000

00
2,000

4,000

6,000

8,000

Wild type

3,000
2,000

0

Mutant

4,000

1,000
10,000 12,000 14,000

H1650: 1,000 cells

8,000
7,000

6,000

2,000

9,000

H1650: 400 cells

7,000

FAM amplitude

9,000
8,000

Mutant

6,000
5,000

Wild type

4,000
3,000
2,000
1,000
00

1,000		3,000		5,000		7,000		9,000		11,000

HEX amplitude

HEX amplitude

0

2,000

4,000

6,000

8,000

10,00012,000 14,000

HEX amplitudeFig. 1. EGFR DNA mutation in cancer cells enriched using the Genesis System with Celselect Slides. 2-D amplitude plot of lung cancer cells spiked in blood followed byDNA extraction shows H1975 (EGFR L858R/T790M), H1650 (exon 19 deletion), and A549 (wild type) with different concentrations of cells collected from the rare cell isolation steps
using the Genesis System with Celselect Slides. High sensitivity of ddPCR Mutation Detection Assays provides precise quantification of T790M, L858R, and exon 19 deletion
mutation in the presence of wild-type and mutant DNA. The black cluster on the plots represents the negative droplets, the green cluster represents the droplets that are positivefor wild-type DNA only, and the blue cluster represents the droplets that are positive for mutant DNA only.

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All trademarks used herein are the property of their respective owner. © 2023 Bio-Rad Laboratories, Inc.
Purchase of Digital PCR and/or Single-Cell NGS Sample Preparation products (the “Products”) from Bio-Rad Laboratories is subject to Bio-Rad Laboratories, Inc. Standard Terms andConditions of Sale, which can be accessed at https://www.bio-rad.com/en-us/terms-conditions. Unless we expressly state otherwise in additional Terms and Conditions, no rights aregranted for you to distribute or resell the Products. Unless we expressly state otherwise in additional Terms and Conditions, no rights are granted for the development or commercializationof diagnostic assays for use with the Products without a license from Bio-Rad. It is the user’s obligation to obtain a commercial license from Bio-Rad for (i) all commercial uses (not justdiagnostic uses) and (ii) sale of assays for use on Bio-Rad’s dPCR and ddSEQ instruments. The Products and/or their use are covered by U.S. and foreign patents and/or pending patentapplications owned by or under license to Bio-Rad Laboratories, Inc. See https://www.bio-rad.com/en-us/trademarks.

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