Despite the advancement of new molecular biology techniques like ddPCR or RNAseq, quantitative PCR (qPCR) is still a golden standard for the quantification of nucleic acids, with applications in diagnostics and research. The method is based on real-time monitoring of amplification of the target DNA allowing its quantification (see basics). Yet, for the method to be quantitative, it has to be done properly.
Amplification efficiency higher than 100% would mean that in each qPCR cycle, more than two copies of the target sequence have been generated.
Is this possible?
How to present the ratio data?
Which are the most probable reasons for a false positive result and how to address the issue?
Researchers often wonder which statistical tests they should use with their qPCR data. Most frequently these questions arise when analysing gene expression data.