Quantitative Real-Time PCR for Gene Expression Analysis

Total RNA was extracted from watermelon or Arabidopsis leaves using an RNA simple Total RNA kit (TIANGEN, Beijing, China). After extraction, the total RNA samples were treated with gDNase, then reverse-transcribed (1 µg per sample) to cDNA using a FastKing RT kit (TIANGEN, Beijing, China). The qRT-PCR assay was performed on a StepOnePlus^TM Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using SYBR^® Premix ExTaq^TM II (2×) kit (Takara, Tokyo, Japan). The gene-specific primers used for the qRT-PCR are listed in Table S1. The qRT-PCR amplification was conducted under the conditions reported by Li et al. [29 (link)]. β-actin or AtActin2 served as the internal control genes for the normalization of gene expression [11 (link),44 (link)]. Relative gene expression was calculated as described by Livak and Schmittgen [45 (link)].