2.4 ⎪ Gene expression analysis using real-time qPCR
RNA extraction, cDNA synthesis, and qPCR were performed as previously
described (Wakao et al. 2014). All primer pairs were confirmed as
having 90–105% amplification efficiency and linear amplification
within their dynamic range in experimental samples using serial
dilutions of cDNA prior to experiments. Relative transcript levels were
calculated by the ΔΔCt method (Livak & Schmittgen 2001) usingPEX4 (AT5G25760) as the internal reference. PEX4 , a
peroxisomal ubiquitin-conjugating enzyme, is an established RT-qPCR
internal reference (Dekkers et al. 2011) and was confirmed in the
RNAseq dataset to have constant expression levels in all conditions and
ecotypes. Primers were designed using Primer3 (Untergasser et al.2012) against the 3´-UTR of each gene to avoid binding to off-target
paralogous genes. A single peak in melt-curve analysis with a unique
melting temperature was observed for each amplicon, verifying that
off-target amplification of paralogous genes was negligible.