PCR, qPCR, dPCR, and ddPCR
TL;DR
PCR amplifies a target DNA region. qPCR measures amplification in real time and is often used for relative abundance. dPCR/ddPCR partitions a sample into many reactions and estimates absolute copy number by counting positive partitions. These methods are fast and sensitive, but only answer questions about targets you designed primers/probes for. Sources: [1], [2]
What it measures
| Method | Measures | Typical oncology use |
|---|---|---|
| PCR | presence/size of an amplicon | clonality check, construct verification |
| RT-PCR | RNA converted to cDNA | fusion transcript or viral transcript detection |
| qPCR | relative or calibrated abundance | gene expression, copy-number estimate |
| dPCR / ddPCR | absolute copies or allele fraction | low-frequency mutation, MRD-like monitoring, CNV |
What it does not measure
- unknown mutations outside the designed assay
- genome-wide context
- transcript isoforms unless specifically designed
- protein abundance
- cell identity
- tissue location
If you do not know what target to ask for, sequencing is usually the better first tool.
Core QC questions
- Are primer/probe sequences documented?
- Is the amplicon specific?
- Are no-template and no-RT controls clean?
- Is RNA integrity acceptable for RT-qPCR?
- Are reference genes stable in this sample type?
- Is amplification efficiency measured?
- For dPCR, are partitions numerous and rain/thresholding handled consistently?
Computational output
| Raw output | Downstream interpretation |
|---|---|
| Ct/Cq | lower value usually means more starting template |
| melt curve | specificity check for dye-based assays |
| droplet/partition counts | absolute copies after Poisson correction |
| standard curve | quantification and efficiency estimate |
Developer notes
- Store primer/probe IDs as structured metadata, not free text.
- Keep raw Ct/Cq values; do not only store normalized fold change.
- Track failed wells and excluded replicates explicitly.
- Record reference genes and normalization method.
- For ddPCR, keep thresholding rules and droplet counts, not just final concentration.
- Never compare qPCR runs without batch, plate, and efficiency context.
References
- Bustin SA, Benes V, Garson JA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009;55:611-622. PMID 19246619. https://doi.org/10.1373/clinchem.2008.112797
- Bustin SA, Benes V, Garson JA, et al. MIQE 2.0: Revision of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments Guidelines. Clin Chem 2025. PMID 40272429. https://doi.org/10.1093/clinchem/hvaf043