Molecular and cell biology

PCR & qPCR interview questions

Nucleic acid amplification, reaction controls and quantitative interpretation.

9 questions, each with a worked answer

What does a no-template control tell you in PCR? Fundamentals Concept
What could cause amplification in a qPCR no-template control? Intermediate Troubleshooting
How would you evaluate primer efficiency and assay specificity? Intermediate Scenario
Why is a reference gene not automatically stable across treatments? Intermediate Concept
How do technical replicates differ from biological replicates in qPCR? Fundamentals Scenario
When would you use absolute rather than relative quantification? Intermediate Scenario
How would you validate primer specificity before using a new qPCR assay for a critical result? Intermediate Troubleshooting
Replicates vary widely only at low template concentration. How would you investigate? Intermediate Troubleshooting
Design a dilution and spike-in experiment to detect PCR inhibition in a difficult sample matrix. Advanced Practical

About PCR & qPCR

PCR copies a chosen stretch of DNA many times over, and quantitative PCR (qPCR) follows that copying cycle by cycle to estimate how much template a sample started with. It is used for gene expression, pathogen detection, copy number and checking cell lines and products. Getting a number is easy.

Getting a trustworthy one depends on primer design, efficiency, reference genes and controls. Guidance such as the MIQE guidelines sets out what a reported qPCR result should state.

Fundamentals: 2
Intermediate: 6
Advanced: 1

What these questions test

Optimize qPCR reactions: Troubleshoot amplification, normalization and control performance.

References

NCBI Bookshelf. National Library of Medicine. ncbi.nlm.nih.gov/books.
academic.oup.com/clinchem/article/71/6/634/8119148.

Practice material written by TheBench.Bio and checked against the references above, reviewed October 23, 2026. Written to practise with, not collected from real interviews.