ddPCR, Digital PCR and qPCR Services by PhD Scientists at Our 20+ Year, FDA-Inspected Lab

Droplet Digital PCR partitions a single reaction into roughly twenty thousand nanoliter droplets, runs PCR in every droplet, and counts which ones light up. Because the readout is counting rather than curve comparison, ddPCR reports absolute copies without a standard curve and resolves small differences qPCR blurs. That is why vector copy number, residual DNA and rare-target questions in regulated programs default to ddPCR here.

When the answer must be absolute rather than relative: vector copy number, transgene copies per genome, residual DNA, rare mutations, or any number a reviewer will read without a standard curve beside it. Partitioning the reaction into droplets turns quantitation into counting, which removes the calibration argument entirely. For expression screens and routine fold-change work, qPCR remains the faster, cheaper answer, and we will tell you which your question needs.

MIQE is the reporting standard that makes a PCR result defensible: documented primer and probe design, efficiency, linearity, LOD and LOQ, reference gene justification and acceptance criteria. Agencies and reviewers increasingly expect it. Our validations and qualifications run to MIQE guidelines on qualified equipment, so the number you file arrives with its evidence attached.

Validated ddPCR assays reporting copies per genome and copies per microgram of DNA across the timepoints your protocol sets, from expansion through long-term persistence, with integration and residual readouts where the program needs them. Data lands in audited tables ready for your regulatory package, and the same accession can feed the flow and cytokine endpoints that run beside PCR in a CGT program.

Whole blood, PBMC, tissues across the biodistribution list, excreta for shedding, FFPE, cell banks and process fluids. Extraction, purification and quantitation run in-house on the same accession as the downstream assay, which keeps chain of custody unbroken and turnaround inside the study window.

A written scope comes back within two business days. Development typically runs two to four weeks depending on target and matrix, validation or qualification follows to the tier your data must defend, and study samples then run batch by batch against your calendar.

Quantitative PCR (qPCR) is similar to traditional PCR, but it can quantify the amount of DNA in the sample. qPCR analysis involves probes and fluorescent dyes that bind to the target DNA. These probes, when excited by a light source, emit different wavelength lights.

Researchers measure the wavelength of each emitted light to determine the amount of target DNA present in the sample. Besides, one may monitor DNA amplification in the actual moment by determining the increase in fluorescence in real time.

PCR amplifies a target so downstream methods can use it; qPCR measures the amplification in real time with fluorescent probes or dyes, which turns amplification into quantitation. Endpoint PCR answers is it there, qPCR answers how much is there, for DNA and RNA targets alike. In this laboratory qPCR carries gene expression and relative quantitation, and hands absolute questions to ddPCR.

Reverse-transcriptase PCR (RT-PCR) is a PCR sub-type that generates cDNA from template mRNA molecules. RT-PCR can detect and quantify RNA viruses. The first step in RT-PCR analysis is reverse transcribing the mRNA template using reverse transcriptase enzyme. This cDNA is then amplified using PCR. RT-PCR is a sensitive and accurate technique to detect RNA viruses present at very low levels.

qPCR can measure gene expression on its own. In qPCR expression analysis, a section of DNA is read to synthesize the mRNA, which then produces the protein of interest. However, in RT-PCR, reverse transcriptase creates cDNA, which is then used in PCR to calculate the mRNA transcripts.

Digital PCR (dPCR) assay is a novel technique for measuring and identifying nucleic acids that can quantify them precisely and detect rare variants. It works by dividing and isolating a DNA or RNA sample into many separate, parallel PCR reactions; some of these reactions have the target molecule (positive) and others do not (negative). Here, a single DNA or RNA molecule can be amplified by a factor of a million or more. During amplification, TaqMan Assays with dye-labeled probes bind to specific sequences to identify targets. When the target sequence is absent, no signal is produced. After PCR analysis, the proportion of negative reactions is used to calculate the exact number of target molecules in the sample, without requiring standards or internal controls.

Digital PCR analysis has many uses and is getting increasingly deployed to:

• Determine the genetic makeup of an organism
• Absolute quantification of DNA and RNA targets
• Perform copy number analysis (CNV)
• Detect rare mutations
• Identify pathogens, their strains, and their viral load
• Analyze proteins with assays that use proximity ligation

Real Time-PCR/qPCR quantification can be absolute or relative. Absolute quantitation determines unknown samples by interpolating their copy number relative to those obtained from a standard curve. Absolute quantitation may be used to study the correlation between viral or gene copy numbers and disease state. Relative quantitation is used to analyze how the activity of genes in a sample is increased or decreased relative to a reference, control sample that has a normal or baseline gene activity, for comparisons of healthy and diseased subjects, for determining genetic or phenotype related differences between samples, or to compare disease or disease treatment sample biomarkers to baseline levels. Both methods involve normalization of gene quantitation by simultaneous or parallel measurement of a housekeeping gene (for example beta actin or GAPDH), the expression levels of which are relatively stable and are not altered by disease state or treatment. This ensures that differences in absolute or relative level of the gene changes observed are from disease state or treatment and not from differences in nucleic acid loading.

There are two approaches to qPCR analysis: dye-based and probe-based. Let us dive deep into each of these qPCR types.

Dye-based qPCR assays

This method measures the fluorescent signals, specifically the binding of the dye to the double-stranded DNA. Initially, the dye shows a weak signal. However, after binding to the double-stranded DNA, the signal increases dramatically. Hence, target sequence amplification increases the fluorescence, which directly relates to the amount of double-stranded DNA in the sample. This approach needs only two primers specific to the sequence, so dye-based qPCR is a rapid way to assess a large number of study samples.

However, dye-based methods can detect all double-stranded DNA present in the sample. This non-specific detection can include primer dimers and non-specific products, leading to inaccurate results. Hence, reaction specificity is verified using the denaturing or melting curve after each qPCR run. Moreover, dye-based qPCR can only quantify one target sequence at a time.

Probe-based qPCR analysis

Probe-based qPCR measures the fluorescent signal of sequence-specific fluorophore-labeled probes. This method is more specific than a dye-based approach and hence is preferred in diagnostic applications. Probe-based qPCR is a multiplexed method that can quantify multiple targets in a single sample reaction, employing a specific fluorophore for each target. Probe designs vary among experiments, though hydrolysis probes are most commonly used.