Table 1.
Candidate reference genes for evaluation across oil palm tissue culture samples.
Table 2.
Gene ontology classification of oil palm candidate reference genes at level 2 using Blast2GO.
Table 3.
PCR amplification efficiencies (Ex) and correlation coefficient (R2) values of oil palm candidate reference genes.
Figure 1.
Mean Ct values of eight candidate reference genes across MA2 and MA8 tissue culture lines.
The range of Ct values across the MA2 and MA8 tissue culture lines were exhibited in boxplot. Ct values of the candidate reference genes were widely distributed between 17 to 30 cycles. Lower and upper ends of the box represent 25th and 75 percentiles, respectively. Horizontal line inside the box is median. Whiskers below and above the box represent minimum and maximum values of the datasets, respectively. Asterisks represent the outliers.
Figure 2.
Determination of the most stably expressed reference genes across MA2 and MA8 tissue culture lines using geNorm software.
Average expression stability values (M) were calculated for each reference gene. The least stable genes with higher M values were excluded in a stepwise manner until the two most stable reference genes were obtained for the tested tissue culture lines.
Figure 3.
Optimal number of reference genes required for accurate and reliable normalization of gene expression data from each set of tissue culture lines as determined by geNorm software.
Calculation of pairwise variation, Vn/n+1 was performed between the two sequential normalization factors (NFn and NFn+1) of reference genes across the same panel of samples. A cutoff value of 0.15 was used to determine whether the inclusion of an additional reference gene has a significant effect on the pairwise variation values.
Table 4.
Ranking of oil palm candidate reference genes using NormFinder analysis.
Table 5.
Ranking of oil palm candidate reference genes according to coefficient of variance (CV) and standard deviation (SD) using BestKeeper analysis.
Figure 4.
Fold change of PD00088 across MA2 and MA8 oil palm leaf explants as determined from the cDNA microarray data analysis.
Microarray datasets from MA2 and MA8 tissue culture lines were analysed using statistical software known as Significance Analysis of Microarrays (SAM), which was developed by Tusher et al., 2001 and deposited as one of the packages under R for window. This software was used to identify differentially expressed genes at each time point between media T527 and T694. Two class unpaired data analysis was selected and the following parameters were applied for each analysis: False Discovery Rate (FDR) of zero, q-value of zero and gene expression ratio cutoff was fixed at 2-fold change.
Figure 5.
Expression profiling of PD00088 across oil palm leaf explants, callus and embryoids using RT-qPCR.
Expression levels of PD00088 in leaf explants (W1, W3), callus and embryoids were normalized with PD000380, PD00569 or combination of both reference genes. Calculation of standard deviation on normalized gene expression level was done using geNorm v3.4 [19]. The error bars represent ± standard deviation (SD).