QuantStudio 3D: absolute DNA quantification by digital partitioning
Thermo Fisher Scientific's QuantStudio 3D is a digital PCR solution that uses compartmentalization to reliably and accurately detect genetic targets in complex mixtures. Unlike traditional real-time PCR systems, this system is based on a binary logic of signal presence or absence in 20,000 independent micro-reactors per chip. This enables absolute quantification without the need for standard curves, significantly reducing biases associated with relative quantification.
The system works by partitioning the DNA or RNA sample converted into cDNA, followed by specific amplification in each well using TaqMan probes. By analysing the number of positive and negative reactions, the system accurately calculates the initial concentration in copies/μL. This approach is particularly effective for detecting low-abundance targets, rare alleles in a wild-type sequence background, or changes in gene copy number.
The tool is particularly useful for experiments where accuracy and reproducibility are critical. Users can improve resolution and accuracy by increasing the number of chips used, creating a "virtual chip" using the QuantStudio 3D AnalysisSuite Cloud online platform. This grouping reduces statistical uncertainty during quantification.
The system also allows subtle variations in gene expression, often invisible to conventional methods, to be measured with a resolution of up to ±10%. Copy number variation experiments can be optimised by fragmenting genomic DNA using restriction enzymes. For experiments on rare alleles, optimisation of amplification conditions, particularly hybridisation temperature, is recommended.
Versatile, compact and intuitive, the QuantStudio 3D fits easily into research laboratories requiring rigorous quantification in fields such as cancer, human genetics, the environment and microbiology.
