Abstract
BackgroundRT-qPCR is a powerful tool for analysing gene expression. It depends on measuring the increase in fluorescence emitted by a DNA-specific dye during the PCR reaction. For relative quantification, where the expression of a target gene is measured in relation to one or multiple reference genes, various mathematical approaches are published. The results of relative quantification can be considerably influenced by the chosen method.ResultsWe quantified gene expression of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in the roots of two black poplar clones, 58-861 and Poli, which were subjected to drought stress. After proving the chosen reference genes actin (ACT), elongation factor 1 (EF1) and ubiquitin (UBQ) to be constantly expressed in the different watering regimes, we applied different approaches for relative quantification to the same raw fluorescence data. The results obtained using the comparative Cq method, LinRegPCR, qBase software and the Pfaffl model showed a good correlation, whereas calculation according to the Liu and Saint method produced highly variable results. However, it has been shown that the most reliable approach for calculation of the amplification efficiency is using the mean increase in fluorescence during PCR in each individual reaction. Accordingly, we could improve the quality of our results by applying the mean amplification efficiencies for each amplicon to the Liu and Saint method.ConclusionsAs we could show that gene expression results can vary depending on the approach used for quantification, we recommend to carefully evaluate different quantification approaches before using them in studies analysing gene expression.
Highlights
RT-qPCR is a powerful tool for analysing gene expression
The major progress of qPCR is that quantification does not have to be done in the plateau phase of amplification, which is a disadvantage of previous quantification methods [1]
When ordering the samples according to ascending Cq values, which maximizes the slopes of the regression lines, the fit was closer than when ordering the samples randomly (R2 = 0.94 for all genes), but the slopes were still low (0.07 for ACT and elongation factor 1 (EF1), respectively, and 0.09 for UBQ), indicating that expression of the genes was not altered by the different drought treatments
Summary
RT-qPCR is a powerful tool for analysing gene expression. It depends on measuring the increase in fluorescence emitted by a DNA-specific dye during the PCR reaction. RT-qPCR is a widely used method for analysing gene expression. It has been developed by combining PCR with fluorescent techniques [1,2]. The major progress of qPCR is that quantification does not have to be done in the plateau phase of amplification, which is a disadvantage of previous quantification methods [1]. PCR can be divided into four major phases: linear ground phase, early exponential phase, log-linear phase and plateau phase [3]. During the log-linear phase, when PCR has reached its optimal amplification period, the amount of fluorescence rises exponentially. When the reaction components become limited, the plateau phase is reached and the fluorescence does not increase anymore [4]
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