Quantitative RT-PCR Analysis of Arabidopsis

RNA for quantitative PCR analysis was obtained from 30-day-old plants. The full rosette was harvested by grinding in liquid nitrogen, and RNA was obtained using the Qiagen RNeasy Plant kit from approximately 100 mg tissue (fresh weight). One μg total RNA was used in 20 μL reverse transcriptase reactions (Roche) using manufacturer’s instructions and a poly dT₁₈V anchored primer at 48°C. cDNA synthesis reactions were halted after one hour by heating at 70°C and then diluted 1:10 (v/v) with pure water. One μL was used as template in a PCR that also included 10 μL 2X SYBR Green mix (Roche), 0.6 μL each forward and reverse primers (10 μM), and pure water for a final volume of 20 μL. Primers DXR-F (5’- A G T A G C G G A T G C G T T G A A G C) and DXR-R (5’- G C G G A T G A A T G A C A A T C T C T A T A T C G) were used in these experiments. cDNA loading in individual reactions was normalized to the levels of APT1 and RP2ls genes, whose sequence and stability under these conditions were previously reported [36 (link)]. Six individual plants were used for each transgenic or wild type line and each biological replicate was analyzed in three technical replicates for each gene of interest or normalizer. Relative fold calculations were performed using the efficiency corrected model [37 (link)].