Fast start dna masterplus sybr green 1 reaction mix

RT-PCR analyses were conducted using SuperScript™ 2 Reverse Transcriptase (Invitrogen), Lightcycler (Roche), and Fast Start DNA MasterPLUS SYBR Green I reaction mix (Roche) as recommended by the manufacturer. Primers used to quantify the expression of reference gene β-actin are 5′-TCT TCC AGC CTT CCT TCC TG-3′ (sense) and 5′-CAC GGA GTA CTT GCG CTC AG-3′ (antisense). Primers used for the quantification of the EBER2 chimeras are 5′-CCG TTG CCC TAG TGG TTT C-3′ (sense) and 5′-ACA GCG GAC AAG CCG AAT A-3′.

After reverse transcription, quantitative real-time RT-PCR (qRT-PCR) for BCAM/Lu was performed using FastStart DNA Master^PLUS SYBR Green I Reaction Mix (Roche) according to the manufacturer’s instruction on LightCycler^® 1.5 (Roche Applied Science). The primer pair for the amplification of BCAM/Lu qRT-PCR was purchased from Qiagen (QuantiTect Primer Assay). After denaturation of cDNA at 95°C for 10 min, temperature cycling (40 cycles) was performed as follows: a denaturation step at 95°C for 10 s, annealing at 58°C for 10 s and an extension step at 72°C for 10 s. Values were normalized to the expression of housekeeping gene glucose 6-phosphate dehydrogenase (GAPDH, Table 1). Cycle thresholds (CT) of the gene of interest (GOI) were compared with those of housekeeping gene (HG) to determine relative expression levels. Relative fold changes between the expression of the GOI in treated and untreated samples were determined by the following equation: fold change = E_GOI^[ΔCTGOI]/E_HG^[ΔCTHG], where E = PCR reaction efficacy and [ΔCT GOI] = (C_{T  untreated} – C^{T  treated})_GOI; [ΔCT HG] = (C_{T  untreated} – C_{T  treated})_HG.

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.