Quantitative PCR (qPCR), also known as real-time PCR, is a laboratory technique that amplifies and simultaneously quantifies DNA in real time. Unlike conventional PCR, which only shows the final amount of DNA, qPCR monitors the accumulation of DNA throughout the reaction using fluorescence.
How qPCR Works
- Reaction Setup
The reaction contains the same components as conventional PCR—template DNA, primers, DNA polymerase, and nucleotides—plus a fluorescent reporter. Two common reporter systems are SYBR Green, a dye that fluoresces when bound to double-stranded DNA, and TaqMan probes, which are sequence-specific fluorescent probes.
- Amplification and Detection
The reaction undergoes the same thermal cycling as standard PCR: denaturation, annealing, and extension. After each cycle, a fluorescence measurement is taken. As more DNA is amplified, the fluorescence signal increases proportionally.
- The Ct Value
The threshold cycle (Ct) is the cycle number at which the fluorescence signal rises above the background level. The Ct value is inversely proportional to the starting amount of target DNA: more starting DNA means fewer cycles are needed to reach the threshold.
- Quantification
Absolute quantification uses a standard curve of known DNA concentrations to calculate the exact amount of target DNA. Relative quantification compares the Ct values of a target gene to a reference gene, determining fold changes in expression.
- Melt Curve Analysis
When SYBR Green is used, a melt curve analysis is performed after amplification. The DNA is slowly heated while fluorescence is monitored. Each DNA product melts at a characteristic temperature, confirming that the correct amplicon was produced and no primer-dimers are present.
Practical qPCR Protocol
Prepare the reaction in a 96-well or 384-well plate. For SYBR Green, mix 5 µL of 2× SYBR Green master mix, 0.5 µL of each primer (10 µM), 2 µL of template cDNA or DNA, and water to 10 µL. For probe-based detection, replace SYBR Green with a 2× probe master mix and add 0.2–0.4 µM of each probe. Seal the plate with optical adhesive film and centrifuge briefly. Run on a real-time cycler with the following program: 95°C for 2 minutes (polymerase activation), then 40 cycles of 95°C for 15 seconds and 60°C for 30–60 seconds (annealing and extension). Collect fluorescence data at the end of each extension step. After cycling, perform a melt curve ramp from 60°C to 95°C if using SYBR Green. For absolute quantification, prepare a standard curve by serially diluting a known template (10^7 to 10^2 copies/µL in 10-fold steps). Plot Ct vs. log copy number and verify the slope is between −3.1 and −3.6 (90–110% efficiency). For relative quantification using the ΔΔCq method, calculate ΔCt = Ct(target) − Ct(reference), then ΔΔCt = ΔCt(treated) − ΔCt(control). Fold change = 2^(−ΔΔCt). Include no-template controls and no-RT controls for RNA-based targets. Run all samples in technical triplicates and discard any with Ct standard deviation > 0.5.
Real-World Application
In gene expression studies for cancer research, RT-qPCR with SYBR Green quantifies mRNA levels of oncogenes such as MYC and tumor suppressors like TP53. GAPDH or ACTB serves as the reference gene. RNA from tumor and adjacent normal tissue is reverse transcribed, and ΔΔCq analysis reveals that MYC is upregulated 8.5-fold in tumors (p < 0.001). Melt curve analysis confirms a single product at the expected Tm, ruling out primer-dimer artifacts.
