PCR Data Analysis & Troubleshooting 4

Question 1

Where should the fluorescence threshold be set for accurate Ct determination?

Accepted Answer

In the exponential phase, above the background

Answer

Setting the threshold in the exponential phase, where amplification is efficient and reproducible, gives reliable Ct values.

Question 2

Why is the plateau phase not used for quantification in real-time PCR?

Accepted Answer

Reagents become limiting, so the final yield no longer reflects the starting quantity

Answer

In the plateau, depleted reagents and product reannealing break the link between yield and starting template.

Question 3

Technical triplicates for one sample give Ct values of 24.1, 25.9, and 24.3. What is the most likely cause?

Accepted Answer

Pipetting error or a well-specific problem

Answer

Replicates are usually expected to agree within about 0.3-0.5 Ct, so one outlier points to pipetting or a well-specific technical problem.

Question 4

What is the main purpose of a passive reference dye such as ROX in qPCR?

Accepted Answer

To normalize for non-PCR fluorescence differences between wells

Answer

ROX corrects for well-to-well differences in volume, optics, and bubbles that are unrelated to amplification.

Question 5

When should the Pfaffl method be used instead of the standard 2^-ΔΔCt method?

Accepted Answer

When target and reference gene efficiencies differ significantly

Answer

Pfaffl includes each gene's measured efficiency, whereas ΔΔCt assumes both are about 100% and equal.

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