2.6. Quantitative Real-Time PCR
Validation of the microarray expression data was performed using quantitative real-time PCR (qPCR). The first strand cDNA prepared from pooled biological replicates of palms at each stage of maturation was used. Two micrograms of total RNA from each sample was used in a reverse transcription reaction using Omniscript Reverse Transcriptase with standard conditions as recommended by manufacturer (QIAGEN). The first strand cDNA synthesis was primed by random hexamer primers. Specific primers were then designed based on the in-house mesocarp transcript database at Sime Darby using the Primer Premier 5.0 software [30]. The qPCR reaction mix consists of 5 μL of a 5X dilution of cDNA, 0.8 μL of forward and reverse primers (10 mM), 10 μL of BIO-RAD iTaq™ Fast SYBR GREEN Supermix with ROX and topped up with distilled water to 20 μL. The PCR cycling conditions were based on optimized conditions suggested by BIO-RAD with 95 °C (1 min) for 1 cycle and followed by 95 °C (15 s) and 55 °C (35 s), for 40 cycles. Relative expression of each transcript was analyzed using qBase Plus 2.0 [31] and normalized against multiple reference genes. In this study, Cyp2 and GRAS were used as previously published [32,33].