ddPCR Gene Expression Assays

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ddPCR Gene Expression Assays

Overview

QX200™ Droplet Digital™ PCR System Brochure

Droplet Digital PCR (ddPCR™) is Bio-Rad's unique digital PCR technology.

  • Unrivaled precision provides absolute quantification of target DNA or RNA molecules without the use of standard curves
  • Addresses the lack of scalable and practical technologies for digital PCR implementation

The QX200 ddPCR system makes this powerful technology accessible to you for unveiling previously unattainable
research levels.

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QX200™ Droplet Digital™ (ddPCR™) System Data
Detection of BRAF V600E Mutation

High sensitivity of the QX200 system allows quantification of BRAF V600E mutation in the presence of wild-type DNA using PrimePCR ddPCR assays. A, 2-D fluorescence amplitude plot generated by QuantaSoft™ software shows triplicate wells of a mixed mutant:wild-type sample. The black cluster on the plot represents the negative droplets, the green cluster represents the droplets that are positive for wild-type DNA only, the blue cluster represents the droplets that are positive for mutant DNA only, and the orange cluster represents the droplets that are positive for both mutant and wild-type DNA. B, 2-D fluorescence amplitude plot shows three replicates of a wild-type-only sample. C, fractional abundance plot shows the percentage frequency (orange markers) of the mutant DNA in a wild-type DNA background. The blue markers indicate the concentration of mutant DNA (copies/µl) and the green markers indicate the concentration of wild-type DNA (copies/µl) in each of three replicate samples. All error bars generated by QuantaSoft software represent the 95% confidence interval.



Detection of Herpes Simplex Virus and β2 Microglobulin

ddPCR enables precise and reproducible detection of herpes simplex virus 1 (HSV-1) and ß2 microglobulin (B2M) targets in a duplex assay. A, 2-D fluorescence amplitude plot shows three merged replicate samples of 10,000 copies/well of HSV-1 duplexed with 20,000 copies/well of Β2M. The black cluster on the plot represents the negative droplets, the green cluster represents the droplets that are positive for HSV-1 only, the blue cluster represents the droplets that are positive for B2M only, and the orange cluster represents the droplets that are positive for both HSV-1 and B2M targets. B, concentration plot shows merged triplicate wells across an HSV-1 sample dilution series from 0 to 40,000 copies in a constant background of 20,000 copies (66 ng human genomic DNA) of B2M. The blue markers indicate HSV-1 copies/µl and the green markers indicate B2M copies/µl. All error bars generated by QuantaSoft software represent the 95% confidence interval.



Detection of Copy Number for
Myelocytomatosis Oncogene and β-Globin

The QX200 system provides superior resolution with copy number (CN) calls from CN1 to CN13. A, 2-D fluorescence amplitude plot shows duplicate wells of a CN6 sample duplexed with myelocytomatosis oncogene (MYC) and β-globin (HBB) assays. The black cluster on the plot represents the negative droplets, the green cluster represents the droplets that are positive for MYC only, the blue cluster represents the droplets that are positive for HBB only, and the orange cluster represents the droplets that are positive for both MYC and HBB targets. B, copy number plot shows samples ranging from CN1 to CN13 with precise duplicate well values. All error bars generated by QuantaSoft software represent the 95% confidence interval. NTC, no template control.



Validation for Next-Generation Sequencing

ddPCR provides nearly 1,000-fold greater sensitivity and an amplification bias–free approach compared to RNA-Seq for validation. A, concentration plot shows a twofold dilution of cDNA generated using Bio-Rad’s iScript™ advanced cDNA synthesis kit for RT-qPCR. Two independent measurement sets were made: one at a high concentration range and one at a lower range, with four points overlapping. B, table shows comparative results between ddPCR and MiSeq. ddPCR detected thousands of copies per well with low-abundance transcripts (TBP and GUSB genes) using an input of 100 ng total RNA, which demonstrates enhanced sensitivity. The MiSeq sequencer detected only single-digit reads per kilobase per million reads (RPKM) for these transcripts. ddPCR, Droplet Digital PCR.



Gene Expression Analysis of Stem Cell Markers

High sensitivity and precision of the QX200 system allow reliable detection of small fold changes with rare transcripts. The TGFβ signaling pathway plays a role in ovarian stem cell regulation, and the addition of TGFβ will increase the number of stem cells in the population, increase the expression of putative stem cell markers, and decrease terminal differentiation markers like E-cadherin (ECAD). Data show expression levels of cKIT, CD133, OCT3/OCT4, and ECAD genes across ten samples with and without TGFβ treatment, normalized to PPIA in this study. Reproducible detection of cDNA copies as low as 0.5–1 target/µl in a 20 µl reaction was achieved. S, sample.


Digital PCR using QX200™ and AutoDG™ ddPCR Systems
Success Stories: Articles and Videos

Success Stories: Articles

Genomic Variation Analysis: Single Nucleotide Polymorphism: Validation of Allele-Specific Expression Predicted by RNA-Seq in Human Brain Specimens
In the context of human brain development, the QX100™ Droplet Digital™ PCR System yields unambiguous results for genotype identification of low-expression genes and provides absolute quantification of allele-specific expression (ASE) without the use of a standard.
Download the article.

Genomic Variation Analysis: Copy Number Variation: DUF1220 Copy Number Variation across Healthy and Diseased Populations
Studying copy number variation (CNV) with the QX100 Droplet Digital PCR System allows the robust identification of genetic factors such as DUF1220 in neurogenetic diseases between individuals with greater accuracy and precision, while effectively validating array CGH data.
Download the article.

Genomic Variation Analysis: Single Nucleotide Polymorphism: Detection of Single Nucleotide Variation Frequencies by Droplet Digital™ PCR
The QX100 Droplet Digital PCR System validated DNA sequencing results of induced pluripotent stem cells (iPSCs). In addition to confirming the high frequency of single nucleotide variations (SNVs), a de novo type of SNV was identified.
Download the article.

Cancer Mutation Analysis: How Droplet Digital PCR Has Revolutionized Noninvasive Plasma-Based Detection of Mutations
Performing Droplet Digital PCR with the QX100 Instrument effectively detected and accurately quantified the EGFR T790M marker in targeted therapies with limited cancer tumor samples.
Download the article.

Viral Quantification: Adeno-Associated Virus Vector Genome Titer Assay
Determining the adeno-associated virus (AAV) vector genome (vg) titer assay is critical in gene therapy applications. The improved sensitivity observed with Droplet Digital PCR (ddPCR™) technology makes it highly suitable for titration of clinical-grade AAV vector preparations.
Download the article.

Gene Expression: Digital Quantification of Potential Therapeutic Target RNAs
Measuring the exact number of long noncoding RNAs (lncRNAs) in subcellular compartments with Droplet Digital PCR (ddPCR) provides insights into their role and mechanism of action.
Download the article.

Third Generation PCR: A Novel Way to Accurately Measure Copy Number Variations in Human Genome
The McCarroll Lab uses Droplet Digital technology to study the distribution and molecular properties of the genome and its influence on gene expression, as well as the genetics underlying schizophrenia and bipolar disorder.
Download the article.

Success Stories: Videos

Droplet Digital™ PCR opens new perspectives in HIV research. Sangamo BioSciences, Inc. is developing HIV therapeutics toward a functional cure using the company’s proprietary zinc finger DNA-binding protein technology.
Download the article.


Professor Alec Morley, a pioneer in digital PCR, and his colleagues in Adelaide have developed tests for detecting acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML) using the unrivaled sensitivity of the QX200 Droplet Digital PCR System.



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Droplet Digital PCR Publications

Browse the growing list of cited peer-reviewed journal articles in Droplet Digital PCR research featuring Bio-Rad products.

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ddPCR Gene Expression Assays enable accurate quantification of small fold changes in gene expression, for e.g. expression differences that are 2-fold or lower, for detecting targets that may be present at low levels of abundance or samples that contain substances that can inhibit a PCR reaction. ddPCR Gene Expression Assays are available as probe or EvaGreen Assays. Protocols are provided for use in One-Step or Two-Step RT-ddPCR protocols. Find and buy ddPCR Gene Expression Assays.

Key Benefits

  • Enables detection of small fold changes in expression
  • Choose from more than 60,000 gene expression assays for human, rat, and mouse
  • Probe or EvaGreen® chemistry

Sample Data
6512-GENE-A
6512-GENE-B
6512-GENE-C

Robust PrimePCR gene expression assays used in ddPCR with EvaGreen provide precise quantification of 18 reference genes. A, 1-D fluorescence amplitude plot shows results of a two-step reverse transcription ddPCR reference assay panel run on human brain RNA. The black clusters on the plot represent the negative droplets and the blue clusters represent the droplets that are positive for the respective reference assay. B, concentration plot shows various gene concentrations of cDNA calculated as copies/µl on a log10 scale. C, calculated RNA concentrations in the original human brain RNA sample for each reference gene. All error bars are generated by QuantaSoft software and represent the 95% confidence interval.

Packaging Options

ddPCR Gene Expression Probe Assays are available with a FAM or HEX fluorophore. Reference assays with the complimentary fluorophore are available. These assays have been designed for maximum specificity and transcript coverage. Assays are available for human, mouse, and rat. Each tube contains 18uM primers and 5uM probe.

Catalog# Product Name Reaction Size
10031252 ddPCR GEX FAM Assay 200
10031253 ddPCR GEX FAM Assay 1000
10031254 ddPCR GEX FAM Assay 2500
10031255 ddPCR GEX HEX Assay 200
10031256 ddPCR GEX HEX Assay 1000
10031257 ddPCR GEX HEX Assay 2500

ddPCR Gene Expression Assays (EvaGreen) consists of unlabeled PCR primer pairs for use with dye-based chemistry such as the ddPCR EvaGreen Supermix. Individual assays are provided as a 20x stock solution. Each tube contains 5uM each primer.

Catalog# Product Name Reaction Size
10031258 ddPCR GEX EvaGreen Assay 200
10031259 ddPCR GEX EvaGreen Assay 1000
10031260 ddPCR GEX EvaGreen Assay 2500

Related Products

Resources

Number Description Options
10048730 PrimePCR™ ddPCR™ Gene Expression Probe Assays Product Insert, Rev A Click to download
10041702 PrimePCR™ ddPCR™ Gene Expression Assays (EvaGreen) Product Insert, Rev A Click to download

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