Watermelon Gene Expression Quantification

Total RNA was extracted using an RNA extraction kit (Axgen, Union City, CA, USA) according to the manufacturer’s instructions. Residual DNA was removed with DNase Mini Kit (Qiagen, Hilden, Germany). One microgram of total RNA was used for reverse transcription using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) following the manufacturer’s instructions. The gene-specific primers for qRT-PCR were designed on the basis of cDNA sequences as shown in Supplemental Table S1 and watermelon β-actin gene was used as an internal control56 (link). The qRT-PCR assay was performed using an iCycler Iq TM Multicolor PCR Detection System (Bio-Rad, Hercules, CA, USA). PCR products were amplified using the Premix ExTaq II (2×) Kit (Takara, Tokyo, Japan). The PCR conditions consisted of denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s and extension at 72 °C for 30 s. The quantification of mRNA levels was performed according to the method of Livak and Schmittgen57 (link).