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.
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.
Bio-Rad’s second-generation digital PCR system provides absolute quantification of target DNA or RNA molecules using EvaGreen or TaqMan Hydrolysis Probes. Unmatched sensitivity and precision for various applications. Read full description
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