Nanodrop 2000 spectrophotometer

Expression analysis was performed as described previously ^{13 (link)}. Briefly, RNA was isolated from 8-day-old seedlings using the Spectrum™ Plant Total RNA kit (Sigma) and quantified with a NanoDrop 2000 Spectrophotometer. 3 μg of total RNA was reverse-transcribed into cDNA with Superscript™ III reverse transcriptase (Invitrogen) and 500 ng of oligodT primer. The resulting cDNAs were diluted 10-fold. 20 μl quantitative PCR reactions were performed using 10 μl Platinum SYBRR Green qPCR SuperMix-UDG kit (Invitrogen), 2 μl of diluted cDNA, 0.5 μl each primer (4 μM), 0.04 μl ROX reference dye, and 6.96 μl H2O. qPCR reactions were performed on a Mx3005P Real-Time PCR System (Agilent) (50°C for 2 mins, 95°C for 5 mins, 50 cycles of 95 °C, 5 secs, 60°C,20 secs, 72°C, 10 secs; 1 cycle of 95 °C 1 min, 55°C 30 secs, 95°C, 30 secs). Relative and absolute quantification were determined against the standard curves using MxPro QPCR software (the standard curves were made by sequentially diluting the synthesized cDNA four-fold until 1/1024; a no reverse transcriptase control was included as a negative control). ACTIN 2 was used as a reference gene. The integrity of the final qPCR products was determined by melting curve analysis. The relative amount of FLC mRNA was normalized to the level of ACTIN 2^{46 (link)}. All experiments were repeated at least three times with similar results.