Cycler iq multi color real time pcr detection system

RNA extracted from young leaves and stalks from 3-week-old plants was treated with DNaseI (Invitrogen, Carlsbad, CA, USA) and reverse transcribed using oligo dT as primer and Superscript reverse transcriptase (Invitrogen, Carlsbad, CA). Actin was used as a reference gene for qRT-PCR analysis. cDNA concentrations in different samples were normalized based on amplification of actin. Primers for 7 potential genes regulating anthocyanin biosynthesis and actin are listed in Additional file 5. To examine the expression of the 8 genes, a PCR mix was prepared with 1 μL cDNA samples as templates in the qRT-PCR assay in the presence of a SYBR Green PCR Master Mix (Invitrogen, USA) and gene-specific primers. The reactions were performed in a BIO-RAD Cycler IQ Multi-Color Real Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The relative levels of the amplified mRNA were evaluated according to Livak and Schmittgen according to the 2^{− ΔΔCt} method using actin gene for normalization [62 ]. Means of 3 different plant replicates were analyzed, using Student’s t-tests to determine significant differences.

From pumpkin fruits, total RNA was extracted and cDNA was synthesized using Superscript reverse transcriptase (Invitrogen, Cartsbad, CA, USA) with oligo dT primers. Concentrations of cDNA were achieved to be equal. qRT-PCR analysis was carried out to analyze the relative expression level of genes from sucrose and carotenoids biosynthetic pathways, using SYBR® Premix Ex TaqTMII (TliRNaseH Plus) (TaKaRa Clontech). The gene specific primers presented in Additional file 1: Table S6. Actin was used as reference gene. BIO-RAD Cycler IQ Multi-Color Real-Time PCR Detection System (BIO-RAD, Hercules, CA, USA) was used to process the PCR mixture under following program: 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. A melting curve was adjusted from 65 °C to 95 °C. Three replications were selected for each fruit from each stage and also for control. The 2^-ΔΔCT method [62 (link)] was used for the quantification of relative expression.