Quality Assurance & Control Flashcards

A qPCR standard curve slope of -3.32 corresponds to what amplification efficiency?

Answer: 100%

Using E = 10^(-1/slope) - 1, a slope of -3.32 gives perfect doubling each cycle, which is 100% efficiency.

What range of amplification efficiency is generally considered acceptable for a validated qPCR assay?

Answer: 90-110%

Efficiencies of 90-110% (slopes of about -3.6 to -3.1) are the standard acceptance range.

A standard curve has a slope of -3.9. What does this indicate?

Answer: Efficiency of about 80%, below the acceptable range

A slope of -3.9 gives about 80% efficiency, which points to a suboptimal assay design or inhibition.

What is the typical minimum acceptable R² value for a qPCR standard curve?

Answer: 0.98 or higher

An R² of at least 0.98 (often ≥0.99) shows the dilution points fall tightly on a linear fit.

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

Answer: Primer-dimers or nonspecific products

Short primer-dimers melt at lower temperatures than the specific amplicon, which produces an extra low-Tm peak.

The limit of detection (LOD) for a clinical PCR assay is commonly defined as the lowest concentration that is:

Answer: Detected in at least 95% of replicates

LOD is usually the lowest concentration detected in at least 95% of replicate tests.

Technical triplicates for one sample show Ct values of 24.1, 24.2, and 26.8. What is the best QC action?

Answer: Treat the outlier as a technical failure and investigate or repeat

Replicate Ct values should normally agree within about 0.3-0.5 cycles, so a 2.6-cycle outlier suggests a pipetting or well error.