Question 5 of 9 Fundamentals Scenario
How do technical replicates differ from biological replicates in qPCR?
Short answer
Technical replicates repeat the measurement of the same biological material and help reveal assay or pipetting variability. Biological replicates come from independently sampled biological units and allow conclusions about variation between those units. Several technical wells from one sample are not several independent biological observations.
Why
A qPCR plate can contain many wells without providing much independent biological evidence. Three wells from one RNA extraction indicate how repeatable the reaction is for that extract. Three independently treated cell cultures or three independently sampled donors provide information about biological variability, assuming the experiment was designed so the units are genuinely independent.
For analysis, summarize technical replicates appropriately after checking their quality and then evaluate the biological units. Otherwise, treating technical wells as independent inflates the apparent sample size and underestimates uncertainty. Replicate structure also depends on whether RNA was extracted independently, whether reverse transcription was repeated and how experimental batches were organized.
A good report makes these layers explicit, not just the number of reactions run.
An example
Four donors are sampled and each cDNA is run in triplicate. The biological sample size is four donors, not twelve PCR wells.
Common mistakes
- Calling multiple wells biological replicates or dropping inconvenient technical readings without a predefined and documented reason
The interviewer might ask next
How would you redesign an experiment if day-to-day extraction variation was suspected?