ddPCR™ Gene Expression Probe Assays
Catalog #Description

10031252
10031253
10031254
10031255
10031256
10031257
12005582
12005583
12005584
12005585
12005586
12005587
12017404
12017425
12017426
12017374
12017427
12017394

ddPCR Gene Expression Assay (FAM), 200 x 20 μl reactions
ddPCR Gene Expression Assay (FAM), 1,000 x 20 μl reactions
ddPCR Gene Expression Assay (FAM), 2,500 x 20 μl reactions
ddPCR Gene Expression Assay (HEX), 200 x 20 μl reactions
ddPCR Gene Expression Assay (HEX), 1,000 x 20 μl reactions
ddPCR Gene Expression Assay (HEX), 2,500 x 20 μl reactions
ddPCR Gene Expression Assay (Cy5), 200 x 20 μl reactions
ddPCR Gene Expression Assay (Cy5), 1,000 x 20 μl reactions
ddPCR Gene Expression Assay (Cy5), 2,500 x 20 μl reactions
ddPCR Gene Expression Assay (Cy5.5), 200 x 20 μl reactions
ddPCR Gene Expression Assay (Cy5.5), 1,000 x 20 μl reactions
ddPCR Gene Expression Assay (Cy5.5), 2,500 x 20 μl reactions
ddPCR Gene Expression Assay (ROX), 200 x 20 μl reactions
ddPCR Gene Expression Assay (ROX), 1,000 x 20 μl reactions
ddPCR Gene Expression Assay (ROX), 2,500 x 20 μl reactions
ddPCR Gene Expression Assay (ATTO 590), 200 x 20 μl reactions
ddPCR Gene Expression Assay (ATTO 590), 1,000 x 20 μl reactions
ddPCR Gene Expression Assay (ATTO 590), 2,500 x 20 μl reactions

For research purposes only.

Description
ddPCR Gene Expression Probe Assays have been designed
for maximum specificity and transcript coverage. Theseassays are available with a FAM, HEX, Cy5, Cy5.5, ROX, or
ATTO 590 fluorophore for human, mouse, and rat genomes.
All the assays can be used with the Droplet Digital™ PCR(ddPCR) Systems.
Ordering Information
ddPCR Gene Expression Probe Assays can be ordered only
online at bio-rad.com/digital-assays.

Reagents and Equipment
For assays using the QX200™ Droplet Generator (catalog#1864002) or Automated Droplet Generator (#1864101):
■

■

■

■

Storage and Stability
ddPCR Gene Expression Probe Assays are stable for
12 months when stored at 4°C protected from light. The assay
mix can be kept at –20°C for long-term storage.
Kit Contents
The ddPCR Gene Expression Probe Assay is a 20x
concentrated, ready-to-use primer-probe mix. Each kit comeswith 200, 1,000, or 2,500 μl of the 20x assay mix (18 μM primers
and 5 μM probe) sufficient for 200, 1,000, or 2,500 x 20 μl
reactions, respectively.

■iScript Advanced cDNA Synthesis Kit for RT-qPCR(#1725037, #1725038)
 or 1–2 targets, ddPCR Supermix for Probes (No dUTP)
F(#1863023, #1863024, #1863025) is recommended or >2 targets, ddPCR Multiplex Supermix (#12005909,F#12005910, #12005911) is recommended
 X200 Droplet Reader (#1864003) or QX600™ Droplet
QReader (#12013328)
PX1 PCR Plate Sealer (#1814000)

For assays using the QX ONE™ Droplet Digital PCR System(#12006536):■■■

iScript Advanced cDNA Synthesis Kit for RT-qPCR(#1725037, #1725038)
 dPCR Multiplex Supermix (#12005909, #12005910,d#12005911)
PX1 PCR Plate Sealer (#1814000)

Refer to the QX200 Droplet Generator Instruction Manual(10031907), QX ONE Droplet Digital PCR System and
QX ONE Software User Guide (10000116512), or AutomatedDroplet Generator Instruction Manual (10043138) for ordering
information on consumables such as oils, cartridges, gaskets,plates, and seals. See Table 1 for a list of fluorophores
compatible with your system.

ddPCR Gene Expression Probe Assays

Table 1. Fluorophore compatibility.

3.

Mix thoroughly by vortexing the tube. Centrifuge briefly
to ensure that all components are at the bottom of thereaction tube. Allow reaction tubes to equilibrate at room
temperature for about 3 minutes.

4.

Transfer the reaction mix from the reaction tubes to theappropriate ddPCR Cartridge as follows:

QX200 Droplet Reader QX ONE ddPCR System QX600 Droplet Reader
FAM

FAM

FAM

HEX

HEX

HEX

Cy5Cy5

CY5.5

Cy5.5
ROX
ATTO 590— For the QX200 Droplet Generator, load 20 μl of eachreaction mix into a sample well of a DG8 Cartridge.
Follow subsequent instructions as specified in
the QX200 Droplet Generator Instruction Manual(10031907)

ProtocolcDNA Synthesis

Make cDNA with iScript Advanced cDNA Synthesis Kit for
RT-qPCR according to the recommended protocol in theproduct insert (10042279).

— For the Automated Droplet Generator, follow
instructions in the Automated Droplet Generator
Instruction Manual (10043138)

cDNA Amount to Include in ddPCR Reaction Mix

Up to 6 μl cDNA (not exceeding the equivalent of 50 ng initialRNA) can be used per ddPCR reaction (20 μl final volume).
Generally, the cDNA resulting from 1 ng RNA is sufficientfor detection of most transcripts. However, dilutions may berequired for abundant transcripts.

— For the QX ONE Droplet Digital PCR System, load
20 μl of each reaction mix into a sample well of a
GCR96 Cartridge. Follow subsequent instructions
as specified in the QX ONE Droplet Digital PCR
System and QX ONE Software Instruction Manual(10000116512)

ddPCR Reaction Mix Setup

1.

2.

Thaw all frozen reaction components to room
temperature. Mix thoroughly by vortexing the tube toensure homogeneity because a concentration gradient
may form during –20°C storage. Centrifuge briefly tocollect contents at the bottom of the tubes.

Thermal Cycling Conditions
Follow instructions for thermal cycling based on the droplet
generator used:■

Prepare samples at room temperature according to therecommendations in Table 2. If multiple samples are to be
assayed using the same target and reference, prepare amaster reaction mix without sample template, dispense
equal aliquots into the reaction tubes, and add the sample
template to each reaction tube as the final step.

■

Table 2. Preparation of the reaction mix for single and multiplex assays.
Volume perReaction, µl

Final Concentration

2x ddPCR Supermix for Probes(No dUTP)

10

1x20x target primers/probe (FAM)

1900 nM primers/
250 nM probe

20x reference primers/probe(HEX)

1900 nM primers/
250 nM probe

cDNA with iScript Advanced
cDNA Synthesis Kit for RT-qPCR

Up to 6Up to 50 ng initial RNA

RNase-/DNase-free water

Variable—Total volume20*—

Component■ or the QX200 Droplet Generator, carefully transfer dropletsF
into a clean 96-well plate. Seal the plate using the PX1 PCR
Plate Sealer at 180°C for 5 sec. Proceed to thermal cycling(see Table 3)
 or the Automated Droplet Generator, remove the droplet
F
plate containing ddPCR droplets from the Automated Droplet
Generator. Seal the plate using the PX1 PCR Plate Sealer at180°C for 5 sec. Proceed to thermal cycling (see Table 3)
 or the QX ONE Droplet Digital PCR System, thermalFcycling is integrated into and sequentially performedby the system itself. Hence, no additional equipmentor sample handling is required for this step. Refer tothe QX ONE Droplet Digital PCR System and QX ONE
Software Instruction Manual (10000116512) for plate setupinstructions. Use appropriate thermal cycling conditions as
specified in Table 3* For the Automated Droplet Generator, prepare 22 μl per well.

© 2022 Bio-Rad Laboratories, Inc.

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10000156269

ddPCR Gene Expression Probe Assays

Other Recommendations
When running technical replicate wells, assemble a common
reaction mix (enough for 1.5x as many wells as you plan to run)with all required components and sample template.

Table 3. Thermal cycling conditions.*Cycling Step

Temperature, °C Time

Numberof Cycles

Hold (QX ONE ddPCR System only)

25

3 min

1

Enzyme activation95

10 min1Denaturation

94

30 sec40

Annealing/extension

55

1 min**

40

Enzyme deactivation98

10 min1

Hold

QX200 or QX600 ddPCR
System (optional)

4

Infinite1

QX ONE ddPCR System(required)

25

1 min

1

Quality Control
ddPCR Gene Expression Probe Assays are free of detectable
DNase and RNase activities. Stringent specifications aremaintained to ensure lot-to-lot consistency.

* For the C1000 Touch Thermal Cycler, use a heated lid set to 105°C and set
the sample volume to 40 µl.
** Check/adjust ramp rate settings to ~2°C/sec.

Data Acquisition and Analysis
Follow instructions for data acquisition and analysis based on
the droplet reader in use:■■■ or the QX200 Droplet Reader, refer to the QX200 Droplet
F
Reader and QX Manager Software Standard Edition UserGuide (10000107223) or the QX200 Droplet Reader andQX Manager Software Regulatory Edition User Guide(10000107224)
 or the QX600 Droplet Reader, refer to the QX600 Droplet
FReader and QX Manager Software Standard Edition
User Guide (10000153877) or the QX600 Droplet Reader
and QX Manager Software Premium Edition User Guide(10000153878)
 or the QX ONE Droplet Digital PCR System, refer to theF
QX ONE Droplet Digital PCR System and QX ONE Software
Instruction Manual (10000116512) and the QX ONE Software
User Guide for Standard Edition (10000116655) or Regulatory
Edition (10000116656)© 2022 Bio-Rad Laboratories, Inc.

310000156269

ddPCR Gene Expression Probe Assays

Visit bio-rad.com/DropletDigitalPCRAssays for more information.

BIO-RAD, DDPCR, DROPLET DIGITAL, QX200, QX600, and QX ONE are trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions. All trademarks used herein are theproperty of their respective owner. © 2022 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 and Conditions of Sale, which can be accessed at https://www.bio-rad.com/en-us/terms-conditions. Unless we expressly state otherwise in additional Terms andConditions, no rights are granted for you to distribute or resell the Products. Unless we expressly state otherwise in additional Terms and Conditions, no rights are granted forthe development or commercialization of diagnostic assays for use with the Products without a license from Bio-Rad. The Products and/or their use are covered by U.S. andforeign patents and/or pending patent applications owned by or under license to Bio-Rad Laboratories, Inc. See https://www.bio-rad.com/en-us/trademarks.

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510-741-1000

10000156269 Ver A (10048730)

22-03630622