Table 1.
Gene description, primer sequences, and PCR efficiency for the selection of hemp reference genes.
Fig 1.
Amplification results for 13 candidate genes using cDNA synthesized from hemp leaf sample to confirm primer specificity and amplicon size.
Fig 2.
Expression level variability of each candidate reference gene to examine all leaf samples (n = 36).
Boxes show the 25th and 75th percentiles, whisker caps represent the minimum and maximum values, lines across the box represent the median Ct-values.
Table 2.
Stability ranking of the 13 candidate reference genes in hemp leaf analyzed by the geNorm program under different stresses.
Fig 3.
Determination of best reference gene number calculated by geNorm pairwise variation (Vn/Vn+1) under different stress treatments in hemp leaf.
Table 3.
Stability ranking of the 13 candidate reference genes in hemp leaf analyzed by the NormFinder program under different stresses.
Table 4.
Stability ranking of the 13 candidate reference genes in hemp leaf analyzed by the BestKeeper program under different stresses.
Data after gene symbols mean Std ± CV%.
Table 5.
Stability ranking of the 13 candidate reference genes in hemp leaf analyzed by the RefFinder program under different stresses.
Fig 4.
Relative expression of target genes in hemp leaf under osmotic stresses using most and least stably expressed reference genes for normalization.
Error bars for qRT-PCR show the standard error of three replicates for EF-1α, TUB, and CHAL, and six replicates for a combination of EF-1α and TUB. The asterisk represents that there is a significant difference in the comparison with the mock treatment by the statistical analysis (P < 0.05) in paired T-tests.
Table 6.
Global ranking of the 13 candidate genes in hemp leaf analyzed by all programs: geNorm, NormFinder, BestKeeper, and RefFinder under different stresses.