Myiq single color real time detection system

Transcript levels of acp genes were measured by real-time PCR using oligonucleotide primers designed with DNAMAN 7.0 software (Lynnon Cooperation). All primer pairs generated one specific product. The expression of gene rpoD was used as the housekeeping control. Cells were harvested at OD₆₀₀ 0.5, 2.0, and 2.6, corresponding to the log, early stationary, and stationary phases, respectively. Total RNA was isolated using RNAqueous kit (Ambion), and genomic DNA was removed by precipitation with LiCl, followed by DNase treatment. cDNA was generated using the High Capability RNA-to-cDNA kit (Ambion), with 1 μg total RNA per reaction. Real-time quantitative PCR was performed in a 96-well plate, using the SensiFAST SYBR^® & Fluorescein kit (Bioline). The reaction mixture contained 500 nM gene-specific primers. Amplification and detection were performed on a Bio-Rad MyiQ single-color real time detection system. Experiments were performed in triplicate for each cDNA preparation. No-reverse transcriptase and no-template controls were included for each assay. Data were analyzed by MyiQ software, and the critical threshold cycle (C_T) was set automatically. The amounts of acp gene mRNA were normalized against rpoD gene using the ΔΔC_T method (Rao et al., 2013 (link)).

All the colour-related uni-transcripts were subjected to real-time quantitative PCR (q-PCR) with specific primers identified by Primer Premier software (Supplementary Table S1 at JXB online). cDNA synthesis and q-PCR were performed as described previously (Qi et al., 2013 (link)). SYBR Green was used for detection of PCR products on a MyiQ Single-Color Real-Time Detection System (Bio-Rad). The actin gene was used as the internal control for normalization of gene expression. At least two independent biological replicates and three technical replicates of each biological replicate for each sample were analysed by q-PCR to ensure reproducibility and reliability. The correlation between expression profiles of colour-related genes measured by q-PCR and RNA-Seq was determined using the R package.