It has been developed by combining PCR with fluorescent techniques. RT-qPCR is a widely used method for analysing gene expression. Accordingly, we could improve the quality of our results by applying the mean amplification efficiencies for each amplicon to the Liu and Saint method. 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. 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. 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. We 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.
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