Data Analysis & Troubleshooting Flashcards

In a qPCR run, Sample A has a Ct of 22 and Sample B has a Ct of 28 for the same target. What does this indicate?

Answer: Sample A had more starting template than Sample B

Ct is inversely related to starting template quantity, so a lower Ct means more initial target.

A standard curve has a slope of -3.32. What amplification efficiency does this correspond to?

Answer: About 100%

Efficiency = 10^(-1/slope) - 1, and a slope of -3.32 gives about 100% (perfect doubling each cycle).

A standard curve gives a slope of -3.9. What is the most likely explanation?

Answer: Efficiency is about 80%, suggesting inhibition or a poor assay design

A slope of -3.9 corresponds to about 80% efficiency, below the usual 90-110% range, which points to inhibitors or a suboptimal assay.

A SYBR Green melt curve shows one peak at 84°C and a second, smaller peak at 74°C. What does the 74°C peak most likely represent?

Answer: Primer dimers or another short nonspecific product

Short products such as primer dimers melt at lower temperatures than the specific amplicon.

In a SYBR Green assay, the no-template control (NTC) amplifies at Ct 37 and its melt peak is well below the target Tm. What is the best interpretation?

Answer: Primer-dimer formation in the NTC

Late NTC amplification with a low-Tm melt peak is typical of primer dimers, not contaminating target.

Using the 2^-ΔΔCt method, a treated sample has a ΔΔCt of -2 relative to the control. What is the fold change in expression?

Answer: 4-fold increase

2^-(-2) = 2^2 = 4, so expression is up 4-fold.

What R² value is generally expected for a reliable qPCR standard curve?

Answer: 0.98 or higher

A well-performing standard curve usually has an R² of at least 0.98, showing tight linearity across dilutions.