Real time RT-PCR as a tool for quantitative Hevea gene expression studies

We describe a real time quantitative rt-pcr (qrt-pcr) approach to measure gene expression of selected latex genes with sybr green i as the reporter dye. in this study,the target genes were the small rubber particle protein (srpp) and farnesyl diphosphate synthase (fpps) while the reference was the 18s ribosomal rna (rrna) gene. as a first requirement,amplification efficiencies of primer pairs specific to target and reference genes were determined by two-step rt-pcr. in the rt (reverse transcription) step,single-stranded cdna was synthesised from latex total rna. then,ten-fold serial dilutions were amplified with gene-specific primers to generate cycle threshold (c t) values for constructing a cdna standard curve for each gene. subsequently,one-step qrt-pcr reactions were set up to generate c t values for the target and reference genes from the same rna sample. a relative quantification method which employs an efficiency-compensated comparative c t approach was used to calculate target gene transcript level relative to the 18s rrna gene.