In quantitative real-time PCR (qRT-PCR) for gene expression analysis, what is the meaning of the Ct (cycle threshold) value, and what comparison method normalizes gene expression across samples?

• A Ct is the total number of PCR cycles; absolute quantification uses a plasmid standard curve
• B Ct is the maximum fluorescence cycle; normalization uses total RNA concentration alone
• C Ct is the cycle number at which fluorescence crosses a threshold; delta-delta Ct (2^-DDCt) method uses a housekeeping gene for normalization ✓
• D Ct is the melting temperature of amplicons; compared to a reference gene's Tm

Explanation

The Ct (cycle threshold) is the PCR cycle number at which the fluorescence signal rises above the background threshold, indicating exponential amplification. A lower Ct indicates more starting template (higher mRNA expression). The delta-delta Ct (2^-DDCt) method normalizes expression: first, subtract the housekeeping gene Ct from the gene of interest Ct (delta-Ct) in each sample; then subtract the reference sample's delta-Ct from the experimental sample's delta-Ct (delta-delta-Ct). The fold change = 2^(-DDCt). Housekeeping genes (GAPDH, beta-actin, 18S rRNA) should be stably expressed across conditions.

Reference: Harper's Illustrated Biochemistry, 32nd ed.

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