Figure 1.
The four gene lists of interest in the microarray experiment, generated from four pair-wise comparisons (or contrasts) of gene expression profiles.
1) gene list 1 is the collection of genes differentially expressed between the dH2O control and 10−7 M LCO-treated plants, for leaves harvested 0 h after foliar spray; 2) gene list 2 is a list of genes differentially expressed between the dH2O control and 10−7 M LCO-treated plants, for leaves harvested 48 h after foliar spray; 3) gene list 3 includes all the differentially expressed genes of the dH2O) control plants during the 48 h time period after foliar spray; 4) gene list 4 consists of genes in the 10−7 M LCO-treated plants that were differentially expressed during the 48 h time period after spray treatment.
Figure 2.
Volcano plots of the EB (Roke algorithm).
A) contrast 1 (time = o h); B) contrast 2 (time = 48 h); C) contrast 3 (treatment = control); D) contrast 4 (treatment = LCO). The volcano plot is a graphical breakdown of the statistical analysis of microarray data. Each point in the plot corresponds to a statistically tested gene. The x-axis is the base 2 logaritm of the fold change, and the Y-axis is the negative base 2 logrithm of the q-value (or adjusted p- value). Thresholds for both the statistical significance (q≤0.05) and the biological significance are highlighted and assembled in the top left and top right corner of the graph, with exception of contrast 1 in which no hit was found.
Figure 3.
Venn diagrams of the output of differentially expressed genes, determined by Cyber-T, LPE and EB (Rocke) algorithms.
A) contrast 1 (treatment = control vs. LCO, time = 0 h), the output is 0 genes; B) contrast 2 (treatment = control vs. LCO, time = 48 h), the output is 147 gene; C) contrast 3 (treatment = control, time = 0 vs. 48 h), the output is 1569 genes; D) contrast 4 (treatment = LCO, time + 0 vs. 48 h), the output is 1260 genes.
Table 1.
Stringency comparison of the three algorithms used in microarray data analysis.
Figure 4.
Heat maps of known genes in gene list 2 showing down-regulated genes.
The heat map depicts the gene expression data of all replicates in a color scheme: red color represents up-regulation and green represents down-regulation; higher color brightness indicates a greater magnitude of differential expression and vice versa. Ideally, the same group of samples should have similar colors.
Figure 5.
Heat maps of known genes in gene list 2 showing up-regulated genes.
A heat map depicts the gene expression data of all replicates in a color scheme: red color represents up-regulation and green represents down-regulation; higher color brightness indicates a greater magnitude of differential expression and vice versa. Ideally, the same group of samples should have similar colors.
Figure 6.
Functional classification of altered gene expression in contrast 2.
Pie charts represent functional classification according to GO implications of A) down-regulated genes; B) up-regulated genes. Fourteen percent of the down-regulated genes and 15% of the up-regulated genes in contrast 2 were related to stress response. Sixteen percent of the down-regulated genes and 2% of the up-regulated genes were related to signaling (signal transduction and transcription). This indicates that under sub-optimal growth temperature (15°C), foliar spray of 10−7 M LCO induced the differential expression of stress related genes and components involved in signaling.
Figure 7.
Heat maps of known genes in gene list 3, showing down-regulated genes.
Figure 8.
Heat maps of known genes in gene list 3, showing up-regulated genes.
Figure 9.
Functional classification of altered gene expression in contrast 3.
Pie charts show functional classification according to GO implications of A) down-regulated genes; B) up-regulated genes. Nine percent of the down-regulated genes and 12% of the up-regulated genes in contrast 3 were related to stress response. Nine percent of the down-regulated genes and 11% of the up-regulated genes were related to signaling (signal transduction and transcription). This indicates that under sub-optimal growth temperature (15°C), the gene expression profile of soybean was dynamic over the period of 48 h; stress-related genes and signaling-related components were active during this time period.
Figure 10.
Heat maps of known genes in gene list 4 showing down-regulated genes.
Figure 11.
Heat maps of known genes in gene list 4 showing up-regulated genes.
Figure 12.
Functional classification of altered gene expression in contrast 4.
Pie charts indicate functional classification according to GO implications of A) down-regulated genes; B) up-regulated genes. Seventeen percent of the down-regulated genes and 17% of the up-regulated genes in contrast 4 were related to stress response. Five percent of the down-regulated genes and 4% of the up-regulated genes were related to signaling (signal transduction and transcription). This indicates that under sub-optimal growth temperature (15°C), the gene expression profile of soybean was dynamic over the period of 48 h following the foliar spray of 10−7 M LCO; stress-related genes and signaling-related components were active during this time period.
Figure 13.
qPCR validation of the microarray data.
Selection of target genes for qPCR validation, among the 147 differentially expressed soybean genes in gene list 2, using the random-stratified method. The 147 genes in gene list 2 were sorted in order of fold change, and divided into 7 equal-sized bins, each of which represented a stratum of fold change magnitude. From each stratum, one gene was randomly selected as a target gene for validation. The details of 7 selected genes in this study is shown in Table 2. A) Correlation of microarray data and qPCR data on a log scale; B) Correlation of microarray and qPCR data based on fold change.
Table 2.
The 7 target genes selected for qPCR validation, plus the housekeeping reference gene β-tubulin [92].
Table 3.
Comparison and correlation between results obtained through qPCR and microarrray, in terms of fold change and p-value (or adjusted p-value).