Quantifying Gene Expression in Citrus Fruit

Available total RNA from the original extractions from peel and newly extracted from whole fruitless were used to undergo gene expression analysis at 126 and 30 DPA, respectively. qRT-PCRs were performed using LightCycler® FastStart DNA MasterPLUS SYBR Green I reaction mix and a LightCycler 2.0 Instrument (Roche, Basel, Switzerland) to determine the relative mRNA levels in each total RNA extraction sample. The fluorescence intensity data was obtained through LightCycler Software version 4.1 and used to calculate the relative expression level of each gene through the ΔΔCt method using CitUBC1 as a housekeeping gene [85 (link)]. Total RNA extraction from Clemenules was used as a control. Specificity of the amplification reactions was assessed by melting temperature profiling of the amplicons yielded by each primer pair. The sequences of the forward and reverse primers and the size of the resulting fragments are listed in Additional file 10.