Practice Test GeeksPCR - Polymerase Chain Reaction Certification Practice Test
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Data Analysis & Troubleshooting Flashcards

7 cards from real PCR practice questions. Tap to flip, then mark Knew It or Still Learning — missed cards come back until you master them.

Read the first 7 Data Analysis & Troubleshooting flashcards as text

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

    Answer: In the exponential phase, above the background

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

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

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

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

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

    Answer: Pipetting error or a well-specific problem

    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.

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

    Answer: To normalize for non-PCR fluorescence differences between wells

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

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

    Answer: When target and reference gene efficiencies differ significantly

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

  6. A reference gene's Ct shifts by 3 cycles between treated and control samples loaded with equal RNA. What should the analyst conclude?

    Answer: The reference gene is not stable under these conditions and is unsuitable for normalization

    Reference genes must stay stable across conditions, and tools such as geNorm or NormFinder are used to choose stable ones.

  7. How is the limit of detection (LOD) of a qPCR assay commonly defined?

    Answer: The lowest concentration detected in at least 95% of replicates

    LOD is usually the lowest target level that is reliably detected, typically in 95% of replicates.

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