Fig. 1. Specificity of RTqPCR and amplicon size. (a) Agarose gel (1.5%) electrophoresis showing amplification of a specific PCR product of the expected size for each gene. (b) Melting curves of one target gene and nine reference genes showing single peaks. M1 and M2 represent D2000 DNA Marker (150 bp-2000 bp) and DNA Marker I (100 bp-600 bp) marker, respectively.
Fig. 3. Average expression stability values (M) and Pairwise variation (V) calculated by geNorm to determine the optimal number of reference genes. The average pairwise variations Vn/Vn+1 was analyzed between the normalization factors NFn and NFn+1 to indicate the optimal number of reference genes required for RT-qPCR data normalization in different samples.
Fig. 4. The expression quantification of ExoGP normalized by TUBβ and GAPDH as internal controls, respectively. Error bars represent standard error of the mean.
Fig. 2. RT-qPCR Ct values for reference genes. Expression data displayed as Ct values for each reference gene in all Ggt and Ggtinfected wheat root samples. A line across the box is depicted as the median. The box indicates the 25th and 75th percentiles, whiskers represent the maximum and minimum values.
Table 1. Candidate reference genes, primers and different parameters derived from RT-qPCR
Table 2. Ranking of candidate reference genes in order of their expression stability as calculated by Norm Finder
Table 3. Statistics results by Best Keeper software for ten se-lected genes based on Ct values
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