Quantifying Gene Expression in Plants

RNA samples were treated with RNase-free DNase (New England Biolabs, Hitchin, UK) to remove remnant DNA, then underwent synthesis of first-strand cDNA by the use of the SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Gene-specific primers for PeTCP genes were designed with the use of Primer Express (Applied Biosystems, Foster City, CA, USA) and are listed in Supplementary Table S1 at JXB online. PeActin4 (5′-TTGTGAGCAACTGGGATG-3′ and 5′-GCCACGCGAAGTTCATTG-3′) and 18S rRNA (5′-TTAGGCCACGGAAGTTTGAG-3′ and 5′-ACACTTCACCG GACCATTCAA-3′) (Lin et al., 2014 (link)) were used as internal control. Each real-time RT-PCR contained 5ng of cDNA, 20mM primers and 12.5ml of SYBR GREEN PCR Master Mix (Applied Biosystems), and water was added to 25ml. Real-time PCR involved use of the ABI 7 500 Real-Time PCR Instrument (Applied Biosystems). For each real-time RT-PCR, each sample was analysed in triplicate. Data were analysed by the use of the Sequencing Detection System v1.2.3 (Applied Biosystems). The software MultiExperiment Viewer was used to construct heatmap representations for expression patterns.