The total RNA was isolated with TRIzol Reagent (Takara, Japan). DNase I (Promega, USA) was used to remove genomic DNA. The total RNA extracted from fruit was used to generate cDNA samples via random priming with Superscript III reverse transcriptase (Invitrogen, Thermo Fisher Scientific, USA).
The cDNA samples were used as templates and were mixed with 10 μM of each primer and SYBR Green PCR Real Master Mix (Tiangen, Beijing, China) for real-time PCR analysis using the ABI 7500 Real Time PCR System and Software 7500 ver. 2.0.3 (Applied Biosystems, USA) as described in the manufacturer’s instructions. The temperature procedure was: 95 °C for 15 min; and 40 cycles of 95 °C for 30 s, 57 °C for 30 s, and 68 °C for 1 min. The fluorescence signal was collected during the elongation at 68 °C of every cycle. The oriental melon 18S rRNA was used as an internal control to normalize small differences in the template amounts. The LOX/18SrRNA, AAT/18SrRNA, SS/18SrRNA and SPS/18SrRNA ratios for all samples were related to the ratio for 5 DAA, which was set to 1. The primers used for real-time qPCR are listed in Additional file1 .
The cDNA samples were used as templates and were mixed with 10 μM of each primer and SYBR Green PCR Real Master Mix (Tiangen, Beijing, China) for real-time PCR analysis using the ABI 7500 Real Time PCR System and Software 7500 ver. 2.0.3 (Applied Biosystems, USA) as described in the manufacturer’s instructions. The temperature procedure was: 95 °C for 15 min; and 40 cycles of 95 °C for 30 s, 57 °C for 30 s, and 68 °C for 1 min. The fluorescence signal was collected during the elongation at 68 °C of every cycle. The oriental melon 18S rRNA was used as an internal control to normalize small differences in the template amounts. The LOX/18SrRNA, AAT/18SrRNA, SS/18SrRNA and SPS/18SrRNA ratios for all samples were related to the ratio for 5 DAA, which was set to 1. The primers used for real-time qPCR are listed in Additional file