Total rna isolation system

Total RNA was extracted from ‘TSH’ and ‘SBT’ pomegranate peels using a Total RNA Isolation System (Tiangen, Beijing, China). The RT-PCR reactions were performed using qPCR SuperMix (TransGen Biotech, Beijing, China) with an Applied Biosystems 7500 real-time PCR system (Applied Biosystems, New York, NY, USA). Expression data were normalized to those of the pomegranate actin gene. Transcription levels were calculated using the cycle threshold (Ct) 2^−ΔΔCt method, according to Livak et al. (2001) [41 (link)]. All primers used in this study are listed in Table S1.

Sampling from 8-week-old plants under the same greenhouse conditions as previously described, qRT-PCR analysis was conducted according to the MIQE guidelines [67 (link), 68 (link)]. Total RNA was isolated using a total RNA isolation system (Tiangen, Beijing, China). First-strand cDNAs were synthesized using a First-strand cDNA Synthesis Kit (Tiangen, Beijing, China). qRT-PCR was performed on a Bio-Rad CFX96TM Real-time PCR System using SYBR Real Master Mix (Transgen, Beijing, China) using the following PCR thermal cycle conditions: pre-denaturation at 95 °C for 30 s; followed by 40 cycles of 95 °C for 5 s, 60 °C for 15 s, and 72 °C for 20 s. 18S RNA (GenBank Accession Number: AJ236016) was used as housekeeping gene. Three biological replicates were performed for each line, and the standard curve method was used for statistical analysis.

Samples stored in the freezer at -80 °C were used for total RNA extraction using a total RNA isolation system (Tiangen, Beijing, China). First-strand cDNAs were synthesized using a First-strand cDNA Synthesis Kit (Tiangen, Beijing, China). The full-length coding sequences of genes were isolated from ‘white winter pearmain’ cDNA. Real-time quantitative reverse transcription (qRT-PCR) analysis was performed according to MIQE guidelines [45 (link), 46 (link)]. qRT-PCR was performed on a Bio-Rad CFX96TM Real-time PCR System using SYBR Real Master Mix (Transgen, Beijing, China) under the following PCR thermal cycling conditions: predenaturation at 95 °C for 30 s; followed by 39 cycles of 95 °C for 5 s, 60 °C for 15 s, and 72 °C for 20 s. MdActin (GenBank Accession Number: AB638619) was used as the housekeeping gene. The sequences of the primers used are listed in Supplementary Table S1. Three biological replicates were performed for each gene, and the standard curve method was applied in statistical analysis.