Ddpcr Quantification
Ddpcr Quantification - Related Videos
09:50
Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina
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2021
This protocol presents how to quantify AAV transduction efficiency in mouse retina using digital droplet PCR (dd-PCR) together with small scale AAV production, intravitreal injection, retinal imaging, and retinal genomic DNA isolation.
07:37
Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR
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Cited by 9 •
2016
A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.
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.
07:06
Isolation and Quantification of Axonal mRNAs Using Porous Membrane Inserts and RTddPCR
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Cited by 1 •
2026
This study presents a robust method to isolate axonal mRNAs using porous membrane inserts, enabling total neuron vs. neurite separation and RNA purification. Combined with RTddPCR, the approach allows absolute quantification of low-copy transcripts, facilitating studies of mRNA transport and local translation with high sensitivity, reproducibility, and broad experimental applicability.
08:45
Duplex Digital PCR for Simultaneous Quantification of Dual Genetic Markers
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2025
In this video, we demonstrate the duplex digital PCR (ddPCR) technique — a modification of the traditional PCR technique that is useful in detecting two different genetic markers simultaneously. A single PCR reaction is partitioned into nanoliter-sized emulsified droplets that are independently amplified, and the detection of differently colored fluorescence amplification signals from the fraction of droplets is used to compute the initial concentration of the target sequences.
FAQs
Absolute concentration is calculated from the proportions of target-positive and target-negative droplets, rather than from comparison with a standard curve. Poisson statistics account for how target molecules are distributed among partitions and convert the observed positive-droplet pattern into an absolute measurement. This supports quantitative results when a conventional calibration approach is limiting.
Partitioning the reaction into thousands of nanoliter-sized droplets makes amplification occur independently in separate reaction spaces. That separation helps distinguish droplets containing the target from those without detectable target after amplification. The resulting positive-versus-negative pattern provides the counting basis for sensitive measurements, including targets present at low abundance in environmental samples.
Fluorescence serves as the readout for target detection after amplification. Droplets are classified according to whether they show a target-associated fluorescent signal, producing positive and negative counts for the statistical calculation. Because the measurement depends on these classifications, the fluorescence pattern directly determines the estimated abundance rather than merely indicating that amplification occurred.
Complex environmental backgrounds and inhibitors can interfere with conventional assays, but ddPCR quantification is described as valuable under those conditions. Its droplet-based measurement can support precise, reproducible detection of low-abundance targets in samples such as water, soil, and wastewater. This makes it useful when matrix complexity could otherwise limit environmental measurements.
An environmental workflow begins with a sample containing the DNA or RNA target, followed by reaction partitioning into droplets and independent PCR amplification. Fluorescence is then used to identify positive and negative droplets, and the counts are analyzed with Poisson statistics. The output is an absolute target concentration for interpreting the sample.
Researchers can apply the method to monitor low-abundance pathogens, antibiotic-resistance genes, and microbial markers in water, soil, or wastewater. These targets connect molecular measurements with pollution assessment, ecosystem surveillance, and public-health decision-making. The appropriate target depends on the environmental question, while the measurement provides a quantitative basis for comparing detected abundance.
In environmental and microbial analysis, ddPCR quantification is especially relevant when the objective is to measure specific targets against complex backgrounds. Its sensitivity and reproducibility support surveillance of environmental change and contamination-related signals, while absolute measurements help characterize target abundance without relying on a standard curve. These outcomes can inform monitoring and public-health decisions.