Table 1.
Description statistics of housekeeping gene candidates.
Fig 1.
Expression level of tested housekeeping genes.
The distribution of Cq (quantification cycle) values of 9 reference genes pooled across 30 samples (15 experimental conditions x 2 time-points) obtained using qPCR. The boxplot marks the median (line) and 25th (lower) and 75th (upper) percentile; x marks the mean; the underlying violin plots show the data distribution for each housekeeping gene. Outliers are depicted as black dots. Data for individual experimental conditions may be found in S4 Fig.
Fig 2.
Individual ranking of housekeeping genes by stability.
Stability values for nine candidate reference genes generated by the following algorithms: A) geNorm, B) NormFinder, C) BestKeeper, and D) a consensus (by rank aggregation). The maxiumum scale for A-C represent the maximum acceptability value for each stability metric.
Fig 3.
Optimal stability ranking of candidate reference genes using geNorm, NormFinder, BestKeeper, and rank aggregation method.
Gene ranks from the three stability algorithms are shown by treatment group. Line colors represent: purple (geNorm), green (NormFinder), yellow (BestKeeper), black (mean rank), and red (consensus rank).
Fig 4.
Calculation of optimal number of housekeeping genes.
Pairwise variation (Vn/n+1) was calculated in all tested samples; all (all conditions together), hor (hormones), nutr (nutrients), phy (physiological stress), pol (pollution), t-l (temperature-light), wou (wounding). The Vn/n+1 values below the 0.15 threshold (dotted line) indicate that n normalization genes are sufficient.
Table 2.
List of recommended reference genes for each of the tested conditions.
Fig 5.
Detection of significant change in gene expression depending on the reference gene choice.
Expression of two heat shock protein genes (Hsp70 and Hsp90) normalized by the two most stable (A: EF1A and GAPDH) and two least stable (B: ARP2/3 and EF2A) reference gene pairs. Statistically different gene expression from ASWP is indicated by * (Student’s t-test; normal distribution, unequal variance; p<0.05).