qPCR relative expression

Paste the Ct table from your instrument. Lattent averages technical replicates, normalises to one or more reference genes, corrects for primer efficiency and gives fold changes with confidence intervals and corrected p-values.

Fold change

How the quantification works

For each biological sample, technical replicate Ct values are averaged. Each gene's relative quantity is computed against the mean Ct of the control group, using that gene's amplification efficiency E (2 = 100%):

RQ = E^{C_t,control − C_t,sample}, NRQ = RQ_target / geomean(RQ_reference)

With 100% efficiency and one reference gene this is exactly 2^−ΔΔC_t (Livak); with measured efficiencies it is the Pfaffl method, generalised to several reference genes (Hellemans et al., 2007).

Statistics are done on log₂(NRQ), where qPCR data are approximately normal. Fold changes are geometric means, intervals are t-based and back-transformed, and each group is compared with the control by Welch's t-test with Holm correction within each gene.

Quality control flags technical replicates more than 0.5 cycles apart, undetermined wells and samples missing a reference gene.

Validated against SciPy and a hand calculation of 2^−ΔΔCt. Validation report.