Rna free dnasei

Total RNA was extracted from the flesh of bottle gourd USA and Hang at the mature stage. RNA-free DNaseI (Fermentas, Waltham, MA, USA) was used to remove DNA contamination for 20 min at 37 °C. Approximately 1 μg of total RNA was reverse transcribed using Oligo(dT)₁₈ and a Fermentas Revert Aid First Strand cDNA Synthesis Kit (Fermentas, Glen Burnie, MD, USA). The reactions were incubated for 30 min at 16 °C, followed by 60 cycles of pulsed reverse transcription at 30 °C for 30 s, 42 °C for 30 s and 50 °C for 1 s; and finally terminated by incubating at 70 °C for 5 min. Relative expression analysis of the mRNA was performed using the ABI Step One Plus™ Real Time PCR System (Applied Biosystems, Carlsbad, CA, USA) and SYBR Green Master Mix (Roche, Berlin, Germany). Quantitative real-time PCR was performed using the following parameters: 5 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. LsH3 was chosen as the endogenous control [39 (link)]. The reactions were performed with three biological replicates, and a melting curve analysis was carried out to verify that only one specific amplification occurred. Comparative expression levels were calculated in the four different samples using the 2^−ΔΔCT method. Primer sequences are provided in Table S1.