Droplet Digital Assays
Droplet Digital Assays - Related Videos
07:37
Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR
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Cited by 10 •
2016
A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.
06:55
Simple Bulk Readout of Digital Nucleic Acid Quantification Assays
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Cited by 5 •
2015
We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.
04:34
Digital Droplet Polymerase Chain Reaction for Indel Mutation Detection in Target Genes
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2025
This video demonstrates the role of digital droplet polymerase chain reaction in identifying indel mutation in a DNA sample. The high precision and sensitivity of ddPCR enable accurate and consistent detection of rare mutations present in a sample, even at low levels. It can identify and quantify indel mutations in different regions of the genome, providing valuable insights into the mechanisms of mutation and the functional consequences of these mutations.
06:38
Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction
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Cited by 4 •
2019
We successfully converted the standard telomere repeat amplification protocol (TRAP) assay to be employed in droplet digital polymerase chain reactions. This new assay, called ddTRAP, is more sensitive and quantitative, allowing for better detection and statistical analysis of telomerase activity within various human cells.
08:39
Quantification of Circular RNAs Using Digital Droplet PCR
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Cited by 8 •
2022
This protocol describes the detailed method of digital droplet PCR (dd-PCR) for precise quantification of circular RNA (circRNA) levels in cells using divergent primers.
FAQs
The key calculation uses the numbers of positive and negative droplets rather than a calibration curve. Poisson statistics model how target molecules are distributed across the partitions and translate the observed pattern into an absolute concentration. This allows the assay to report how much target is present directly from endpoint classifications, which is especially useful when precise quantification is needed.
Endpoint fluorescence provides a signal for identifying which droplets contain the target. Each droplet can therefore be classified as positive or negative after the reaction reaches its endpoint. Counting these separate outcomes preserves information from thousands of reactions instead of averaging the sample into one measurement, helping reveal low-abundance targets and rare genetic variants.
Unlike approaches that depend on a standard curve, Droplet Digital Assays derive concentration from partition counts and Poisson statistics. This reduces reliance on curve-based calibration and contributes to resistance against many sample and amplification differences. The distinction is important when researchers need absolute quantification of low-abundance targets or comparable measurements across biological experiments.
Their resistance to many sample and amplification differences is an important performance characteristic. This makes the method valuable when such differences could complicate interpretation of target measurements. The benefit is especially relevant to biological questions involving low-abundance biomarkers, rare genetic variants, or repeated measurements over time, where reliable quantification supports clearer comparisons.
A basic workflow starts by partitioning the biological sample into nanoliter-scale droplets. After the reactions reach an endpoint, fluorescence identifies droplets containing the target. The positive and negative counts are then analyzed with Poisson statistics to calculate absolute concentration. This sequence connects physical partitioning, signal classification, and statistical interpretation within a single measurement workflow.
These assays can quantify nucleic acids, detect rare genetic variants, measure pathogens, and analyze low-abundance biomarkers. Their high sensitivity and precise absolute measurement make them suitable for research questions where the target may be scarce in a biological sample. They can also support longitudinal studies, in which measurements are collected repeatedly to examine biological changes over time.
Longitudinal studies benefit from measurements that can be compared across multiple time points. By providing absolute concentrations from positive and negative droplet counts, these assays offer a consistent basis for following nucleic acids, pathogens, rare variants, or biomarkers over time. Their resistance to many sample and amplification differences further supports their use in repeated biological measurements.