Abstract
Quantitative real-time polymerase chain reaction (qPCR) is routinely conducted for DNA quantitative analysis using the cycle-threshold (Ct) method, which assumes uniform/optimum template amplification. In practice, amplification efficiencies vary from cycle to cycle in a PCR reaction, and often decline as the amplification proceeds, which results in substantial errors in measurement. This study reveals the cumulative error for quantification of initial template amounts, due to the difference between the assumed perfect amplification efficiency and actual one in each amplification cycle. The novel CyC* method involves determination of both the earliest amplification cycle detectable above background (“outlier” C*) and the amplification efficiency over the cycle range from C* to the next two amplification cycles; subsequent analysis allows the calculation of initial template amount with minimal cumulative error. Simulation tests indicated that the CyC* method resulted in significantly less variation in the predicted initial DNA level represented as fluorescence intensity F0 when the outlier cycle C* was advanced to an earlier cycle. Performance comparison revealed that CyC* was better than the majority of 13 established qPCR data analysis methods in terms of bias, linearity, reproducibility, and resolution. Actual PCR test also suggested that relative expression levels of nine genes in tea leaves obtained using CyC* were much closer to the real value than those obtained with the conventional 2-ΔΔCt method. Our data indicated that increasing the input of initial template was effective in advancing emergence of the earliest amplification cycle among the tested variants. A computer program (CyC* method) was compiled to perform the data processing. This novel method can minimize cumulative error over the amplification process, and thus, can improve qPCR analysis.
Highlights
Our study indicated that this error in the quantification of initial template amount could increase as amplification proceeded; this was due to both the difference in the actual template amount and estimates which were based on the assumption of perfect amplification efficiency
The conventional and widely employed threshold cycle (Ct) method requires assumed perfect amplification efficiency [13], which rarely occurs in practice [29,33]; this leads to an inaccurate estimate of initial template amounts of both the genes of interest and of reference genes
Our study revealed the existence of cumulative error in the Quantitative real-time polymerase chain reaction (qPCR) data processing methods which require the assumption of perfect amplification efficiency
Summary
Nucleic acid samplesIn this study, DNA segments used for quantification assays were either the plant genes or partial gene sequences present in pEASY-T1 plasmids (GenBank EU233623.1). The plasmids previously constructed in this lab each contained a PCR cloned gene of our interest, such as GREEN FLUORESCENT PROTEIN (GFP) (GenBank, U87973.1) (717 bp), NEOMYCIN PHOSPHOTRANSFERASE (npt II) (GenBank, ABW88015.1) present in the pEASY-T1. Isolation of these plasmids from Escherichia coli strain DH5α was performed with Axygen Plasmid Midi Kit (Corning China, Shanghai) using the manufacturer’s instructions. These plasmids were used to test the effects of initial template amounts, amplified product sizes, and primer mismatches on outlier cycle emergence in a PCR procedure. Quality and quantity of DNA and RNA samples were determined using both agarose gel electrophoresis and the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA)
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