Real-Time PCR, RT-PCR, and Nested PCR

Watching amplification

Real-time PCR (qPCR) measures fluorescence in every cycle - from a probe or an intercalating dye (see Probe Chemistries) - so you see product accumulate ¹. The amplification plot is fluorescence against cycle: flat baseline, a steep exponential rise, then plateau.

Threshold and Cq

The software sets a threshold above background, in the exponential region. The cycle at which a sample’s curve crosses it is the quantification cycle (Cq), also called Ct ¹.

• More starting template, earlier Cq. At 100% efficiency, each cycle earlier means twice as much template: a 3-cycle difference is 2³ = 8-fold; 3.3 cycles is about 10-fold.
• Late Cq (around 35-40) means very little target, or nonspecific signal; assays define a cutoff.
• No crossing: not detected - valid only if the internal control amplified.

Because Cq is read in the exponential phase, it reflects starting amount in a way endpoint yield can’t (see PCR Principles).

Absolute quantification

A standard curve from calibrators of known concentration - usually a 10-fold dilution series - plots Cq against log copies ¹. Unknowns are read off the line.

• Slope gives efficiency: E = 10^(-1/slope) - 1. A slope of -3.32 is 100% (perfect doubling); 90-110% (about -3.6 to -3.1) is the usual acceptable range.
• R² near 0.99 shows a linear fit.
• Viral loads (HIV-1, HBV, HCV, CMV) report copies or international units per milliliter this way, calibrated to WHO international standards where they exist.

Relative quantification

To compare a target between samples, normalize to a reference gene measured in the same sample ¹:

1. ΔCq = Cq(target) - Cq(reference), for each sample.
2. ΔΔCq = ΔCq(test) - ΔCq(calibrator).
3. Fold change = 2^-ΔΔCq (assuming near-100% efficiency for both).

Worked example: target Cq 26 and reference 20 in a treated sample (ΔCq 6); target 28 and reference 20 in the control (ΔCq 8). ΔΔCq = -2, so the target is 2² = 4-fold higher in the treated sample.

BCR::ABL1 monitoring is a clinical cousin: the fusion transcript is normalized to ABL1 and converted to the International Scale (see the CML lesson in the applications course).

Melt confirmation

With SYBR-type dyes, finish every run with a melt curve: one sharp peak means one product; extra or lower peaks mean primer dimers or nonspecific products ².

RT-PCR

RNA targets need reverse transcription to cDNA first ¹:

Include a no-RT control to show signal isn’t from contaminating DNA, and use exon-spanning primers when the target is mRNA (see Transcription and RNA Processing).

Nested PCR

Nested PCR runs two rounds: an outer primer pair, then an inner pair inside the first product ¹. The second round both boosts sensitivity and adds specificity, since off-target first-round products rarely carry both inner sites. Hemi-nested PCR reuses one outer primer.

The price: first-round tubes full of amplicon must be opened and transferred - a major contamination risk. Real-time and closed-tube methods have largely replaced nested PCR in routine diagnostics, though it survives in some sensitive pathogen assays.

What to take from this

qPCR reads fluorescence every cycle; Cq is where a curve crosses the threshold, and each cycle earlier means twice the template. A standard curve gives absolute copies, with efficiency = 10^(-1/slope) - 1 (slope -3.32 = 100%; accept 90-110%). Relative quantification normalizes to a reference gene: fold change = 2^-ΔΔCq. RT-PCR adds reverse transcription in one or two steps, with a no-RT control. Nested PCR is sensitive but risky because tubes are opened between rounds.