RT-qPCR Transcriptome Analysis Protocol

Total RNA was extracted with the RNeasy Plant Kit (Qiagen) from samples homogenized in liquid nitrogen with mortar and pestle (2 × 3 repetitions). RNA was treated with rDNase from NucleoSpin RNA Plant kit (Machery-Nagel). cDNA was synthesized using M-MLV Reverse Transcriptase (RNase H Minus, Point Mutant, Promega), oligo dT primers and the Protector RNase Inhibitor (Roche Applied Science). cDNA (20x diluted) was mixed with the LightCycler 480 DNA SYBR Green I Master (Roche Applied Science) and 500 nM of respective primers to a final volume 10 μl. The RT-qPCR was performed with the Light Cycler 480 (Roche Applied Science). qPCR program was set on initial denaturation (5 min, 95°C), followed by 45 cycles of primer denaturation (10 s, 95°C), annealing (10 s, 60°C) and elongation (10 s, 72°C). Relative content of RNA was calculated according to Hellemans et al. (2007 (link)). AtUBQ10 was used as the reference gene.
Primers were designed according to sequences retrieved from TAIR database (Lamesch et al., 2012 (link)) using Primer3Plus program (Untergasser et al., 2007 (link)). The quality of primers was verified by AlleleID (PREMIER Biosoft; Apte and Singh, 2007 ) and the probability of folding secondary structures was predicted in mfold (Zuker et al., 1999 ). Primer sequences are shown in Table S1.