qPCR result for Technical replicates
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Entering edit mode
23 months ago
Maria17 ▴ 40

Hi All,

Currently, I'm working on a qPCR assay. I have done two qPCR tests with the same protocol from the same sample but I've gotten different results.

Based on other experiments, I know that the results of technical replicates are not the same. Would I expect to get the same result or would I expect a different result?

Any recommendations or solutions would be appreciated.

Thanks

Maria

assay PCR qPCR • 734 views
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Entering edit mode
23 months ago
a_bis ▴ 40

Hi! It's almost impossible (and a little suspicious) to get exactly the same result in technical replicates (perhaps you pipetted a slightly different amount of cDNA the second time, the enzyme was a little older the second time, it could be a hundred things). The point of the technical replicates is to see what your inter-replicate variation is like, and if it's consistently small between samples. If the differences you see within each sample's replicates are bigger than the differences you're seeing between, say, your test, positive and negative control samples, perhaps you need to design new primers for your target, pick different positive/negative controls, or redesign your cycling conditions. I hope this is what you were looking for!

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Entering edit mode
23 months ago
Mensur Dlakic ★ 27k

This is not a bioinformatics question.

The reproducibility of experiments depends on the quality of equipment (for example, pipette calibration, tube heat conductivity, cycler ramping), experimental technique (some people pipet more reproducibly than others) and experimental setup (easier to be reproducible when setting up many reactions in batch and in larger volume than a single reaction in a small volume).

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