RNA Extraction and Quantitative RT-PCR

A frozen powder of 100 mg of D. carota leaves from plants transferred to soil and grown in the greenhouse for 6 months was used for total RNA extraction using TRIzol^® reagent (Invitrogen) and following the manufacturer’s instructions. For cDNA synthesis, 2 μg of total DNA-free RNA was mixed with 1mM of oligodT primer and Impron II reverse transcriptase (Promega). The expression of the DXS and DXR transgenes was estimated by RT-PCR using primers qDXRF, qDXSF and qeGFP R (Supplementary Table S1). Quantitative RT-PCR (qRT) experiments were performed in a Stratagene Mx3000P thermocycler, using SYBR Green double strand DNA binding dye as described previously (Stange et al., 2008). Specific primers for carrot genes were designed targeting the 5′ UTR of PSY1 ({"type":"entrez-nucleotide","attrs":{"text":"AB032797","term_id":"6002458","term_text":"AB032797"}}AB032797) and PSY2 ({"type":"entrez-nucleotide","attrs":{"text":"DQ192187","term_id":"79154729","term_text":"DQ192187"}}DQ192187) and the coding sequence of the 18S gene, selected as the normalizer (Supplementary Table S1). Final data were obtained introducing fluorescence results in the equation described by Pfaffl (2001). Each qRT-PCR reaction was performed with three biological replicates and each sample was analyzed in duplicate (technical replicate). In all cases, the reaction specificities were tested with melting gradient dissociation curves and electrophoresis gels. To test for significant differences in gene expression, results were analyzed using the General Linear Models option in the statistical software package Graphpad Prism. The one and two tailed Student t-test (p < 0.05, confidence interval 95%), were used.