Table 1.
Primers and amplicon characteristics for candidate internal control genes, AGO2 and RdR6.
Figure 1.
Expression levels of candidate reference genes in healthy and virus-infected leaf tissue samples of Nicotiana benthamiana.
Values are given as the cycle threshold (Ct, mean of triplicate samples) and are inversely proportional to the amount of template. Global expression levels of the different genes tested are shown as the 25th and 75th quartiles (horizontal lines), median (central horizontal line) and whiskers. Whiskers go either from the minimal to maximal value or, if the distance from the first quartile to the minimum value is more than 1.5 times the interquartile range (IQR), from the smallest value included within the IQR to the first quartile. Outliers, values smaller (Min) or larger (Max) than 1.5 times the IQR, are indicated. Genes are in order from the most (lower Ct, on the left) to the least abundantly expressed (higher Ct, on the right).
Figure 2.
Expression stability of the candidate reference genes analysed by geNorm.
(A) Average expression stability values (M) following stepwise exclusion of the least stable reference genes across all treatment groups. A lower M value indicates more stable gene expression. (B) The optimal number of reference genes required for effective qRT–PCR data normalisation. The pairwise variation (Vn/Vn+1) was analysed between the normalisation factors NFn and NFn+1 using geNorm software to determine whether inclusion of an additional reference gene adds to the stability of the normalisation factor.
Figure 3.
Expression stability of the candidate reference genes analysed by NormFinder.
Error bars represent ± standard errors.
Table 2.
Ranking of the candidate reference genes according to their stability value using geNorm, NormFinder and BestKeeper analyses.
Table 3.
Descriptive statistics of the fourteen candidate reference genes based on their cycle threshold (Ct) values as calculated by BestKeeper.
Figure 4.
Relative quantification of AGO2 and RdR6 expression in Nicotiana benthamiana infected with different viruses, with PP2A, F-BOX and L23 in combination as reference genes.
Error bars represent mean standard error calculated from three biological replicates. Normalisation factors were calculated as the geometric mean of the expression levels of the three most stable reference genes (F-BOX, PP2A and L23) as determined by geNorm analysis. A control mock-inoculated sample was used as the calibrator ( = 1).