Evaluation validation of a qPCR curve analysis method and conventional approaches

Abstract

Abstract Background Reverse Transcription quantitative polymerase chain reaction (RT-qPCR) is a sensitive and reliable method for mRNA quantification and rapid analysis of gene expression from a large number of starting templates. It is based on the statistical significance of the beginning of exponential phase in real-time PCR kinetics, reflecting quantitative cycle of the initial target quantity and the efficiency of the PCR reaction (the fold increase of product per cycle). Results We used the large clinical biomarker dataset and 94-replicates-4-dilutions set which was published previously as research tools, then proposed a new qPCR curve analysis method——CqMAN, to determine the position of quantitative cycle as well as the efficiency of the PCR reaction and applied in the calculations. To verify algorithm performance, 20 genes from biomarker and partial data with concentration gradients from 94-replicates-4-dilutions set of MYCN gene were used to compare our method with various publicly available methods and established a suitable evaluation index system. Conclusions The results show that CqMAN method is comparable to other methods and can be a feasible method which applied to our self-developed qPCR data processing and analysis software, providing a simple tool for qPCR analysis.

Keywords

• Reverse transcription quantitative polymerase chain reaction
• Curve analysis method
• CqMAN
• Performance indicators