Evaluation of a homemade SYBR® Green I reaction mixture for real-time PCR quantification of gene expression

Real-time PCR is an accurate method that can be used for the quantification of specific DNA molecules. Here we provide a protocol for SYBR(R) Green I in real-time PCR applications using plastic reaction tubes. We report that SYBR Green I is alkali labile and once degraded inhibits the PCR. In our optimized protocol, diluted aliquots of SYBR Green I remain stable for at least two weeks. We also evaluated different cDNA synthesis protocols for the quantification of multiple genes from the same cDNA preparation. The best result vas obtained with cDNAs synthesized by OmniScript(TM) reverse transcriptase from 2.5 mug total RNA using oligo d(T)(18) primers. The cDNA reactions could be diluted 1:25, allowing the quantification of up to 125 different medium expressed genes of Arabidopsis. Extension times ranged between 20 and 40 bp/s for accurate quantification of PCR products up to approximately 400 bp in the Rotor-Gene 2000 system. Using our optimized real-time PCR protocol, the reproducibility and amplification efficiency vas high and comparable to a commercially available SYBR Green I kit. Furthermore, the sensitivity allowed its to quantity 10-20 copies of mRNA and dsDNA. Thus, the protocol eliminates the need for expensive pre-made kits.