3.4. Validation by Quantitative Real-time PCR (qPCR)
To validate the expression patterns observed using the Oil Palm mesocarp microarray platform, qPCR was performed to compare the expression level of selected candidates. qPCR serves as a validation tool due to its greater precision and better dynamic range compared to microarray [42]. A total of 12 candidates were randomly selected from the oil palm microarray experiment based on three categories of expression: high, medium and low. The selected genes included those involved in various biological pathways, including some transcripts with unknown function. Primer sets were designed based on published transcriptome sequencing data. The efficiencies of the primer sets were determined and the specificity of the primer sets were analyzed based on dissociation curves (Supplementary Data—qPCR primer efficiency). The qPCR results were normalized using in-house validated oil palm reference genes [32] and show good correlation (Pearson correlation) with the expression profiles in the microarray experiment (R2 = 0.729, p-value < 0.01). The expression profiles generally agreed with the results obtained from oil palm microarray hybridization (Figure 7) with increasing and decreasing trends over the time points in a similar pattern to the microarray expression profile over the same time points. In some cases, the signal magnitude of the microarray experiment was different from the qPCR results, with qPCR expression showing more dramatic changes between time points. This possibly reflects the greater dynamic range of detection [42,43] of qPCR and microarray platforms, which have been reported to generally underestimate the relative changes of expression level in samples tested [44]. However, the overall correlation observed between microarray and qPCR analysis indicates the utility of the oil palm custom mesocarp array to identify expression patterns and differential gene expression between samples.