Human deoxyribonucleic acid (DNA), computer illustration.

Droplet Digital PCR (ddPCR)

Labcorp now offers ddPCR technology to allow nucleic acid quantification with ultrasensitivity and high precision using Bio-Rad’s QX200 System. ddPCR enables absolute quantification of molecular targets that are undetectable with traditional techniques.

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How does ddPCR work?

The ddPCR technique is based on the partitioning of nucleic acid samples into tens of thousands of microdroplets of defined volume in a water-oil emulsion. Amplification of target nucleic acid sequences is carried out within each microdroplet using a similar workflow and reagents as used in a standard TaqMan® probe-based assay. Based on the fluorescence detection after amplification, each droplet is counted and scored as either PCR-positive or PCR-negative. The target DNA/RNA template concentration (copies/µl) in the original sample is quantified using a Poisson distribution.1,2

Applications of ddPCR:

  • Quantify the number of integrated CAR sequences in cell therapy samples
  • Quantify the number of CAR sequences in the blood/tissues of study subjects over time
  • Quantify the number of integrated overexpression constructs in cells/tissues after gene delivery
  • Detect and quantify circulating tumor DNA (ctDNA) in blood
  • Quantify therapeutic bacteria in tissues/tumors
  • Assess tumor mutational burden by quantifying microsatellite instability
  • Detect rare mutations in oncogenes or tumor suppressor genes such as BRCA1, BRCA2, PT53, HER2, RAS, RB1, APC, ErbB2, PI3KCA, MYC or CCND1
  • Determine copy number variation among samples
  • Measure small fold differences in gene expression levels among samples/treatments
  • SNP genotyping
  • Viral load/titer analysis
  • Microbial/pathogen detection
  • Next-generation sequencing library quantification
  • Verify/quantify genomic editing events (NHEJ and HDR from CRISPR) 

Advantages of ddPCR over qPCR:

  • Absolute quantification of nucleic acids without the need for standard curves or serial dilutions
  • ddPCR offers higher accuracy as it generates tens of thousands of partition PCR reactions for each sample as compared to qPCR
  • Increased precision, robustness and sensitivity in detecting less abundant target nucleic acid copies
  • Monitoring of subtle changes in target nucleic acid levels among samples that cannot be detected with real-time PCR
  • Simplified quantification without the need for references or calibration standards
  • Small sample volume requirement

ddPCR FAQs

  • Why should I use ddPCR instead of qPCR?

    ddPCR can measure absolute quantities of nucleic acid sequences per sample volume without extrapolations from standard curves or reference standards. It can detect small fold changes in very low sample volumes that may not be detected by qPCR. ddPCR results are more accurate, sensitive and precise than qPCR results due to the thousands of data points for each sample.

  • What types of nucleic acids does ddPCR detect

    ddPCR can detect any types of DNA or RNA sequences for which primers and probes can be constructed, such as ctDNA, cfDNA/RNA, mRNA, rRNA, snRNA, snoRNA, miRNA or siRNA.

  • What types of samples can be used with ddPCR?

    Any sample that contains DNA or RNA sequences can be used in ddPCR, such as cell and tissue lysates, tissues, biological fluids (blood, cerebrospinal fluid, tears, urine, saliva, etc.), cell supernatants or synthetic nucleic acid products.

  • Does ddPCR take longer than qPCR?

    Like qPCR, a typical run takes about four hours. The ddPCR workflow requires an extra step to create the tens of thousands of nanoliter-sized oil microdroplets. However, the extra time for this step is negated by the simplified digital analysis of the ddPCR reader.

  • In what format should I ship my samples for ddPCR analysis?

    Samples consisting of extracted/isolated/purified DNA/RNA can be run and analyzed with the Bio-Rad QX200 ddPCR System immediately. Cell supernatants and cell/tissue lysates will require DNA or RNA extraction/isolation/purification before the ddPCR assay can be performed. To ensure the safety of the Labcorp laboratory staff, tissues, biological fluids and cell supernatants will require mandatory pathogen testing before handling in the Labcorp laboratories. Additionally, all human-sourced materials (tissues or body fluids), microorganisms or viruses, products created in biological systems with potential biohazard contamination, and recombinant and/or synthetic nucleic acid molecules require the completion of a Biosafety Registration Form and subsequent approval by the Labcorp PCO Biosafety Committee prior to the handling of the materials.

  • How should I prepare cell or tissue samples before shipping them?

    If you are not extracting/isolating/purifying the DNA/RNA prior to shipping the samples for ddPCR, Labcorp PCO recommends protecting/stabilizing/preserving the nucleic acids in cell and tissue samples with a nucleic acid preservation solution (such as RNAlater®, DNA/RNA ShieldTM, or RNAprotect®) before freezing and shipping them.

  • What temperature should I use to ship my samples for ddPCR analysis?

    All samples should be shipped frozen in an insulated box with dry ice.

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References
 

1. QX200 Droplet Digital PCR system. BIO-RAD. https://www.bio-rad.com/en-us/life-science/digital-pcr/qx200-droplet-digital-pcr-system.

2. Taylor SC, Laperriere G, Germain H. Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Sci Rep. 2017;7:2409. doi:10.1038/s41598-017-02217-x.

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