Tri reagent rna clean up kit

Total RNA was isolated using FavorPrep Tissue Total RNA Extraction Mini Kit (Favorgen, Ping-Tung, Taiwan). Afterwards, Tri-Reagent RNA clean-up kit (Favorgen, Ping-Tung, Taiwan) was applied, and complementary DNA (cDNA) was synthesised using the iScript cDNA synthesis kit (Bio-Rad, USA). A PCR was performed using the KAPA2G ReadyMix PCR kit (Kapa Biosystems, Cape Town, South Africa)⁵⁷ . The gene-specific primer sequences are presented in Supplementary Table 1.

Total RNA was extracted from the flavedo of fruit at the time of sampling from the tree (0 d, on-tree fruit), after 4 d for the ethylene, 1-MCP+ethylene, and control groups, and after 28 d storage at either 5, 10, 15, 20, or 25 °C. To remove genomic DNA contamination from the extracted RNA, treatment with DNase I (Nippon Gene, Tokyo, Japan) was carried out, followed by clean-up with FavorPrep after using a Tri-Reagent RNA Clean-up Kit (Favorgen Biotech. Co., Ping-Tung, Taiwan). For all treatments, 2.4 µg of clean RNA was reverse-transcribed to cDNA using a TaKaRa RNA PCR™ kit. Gene-specific primers were designed using the Primer3 software (v.0.4.0, http://bioinfo.ut.ee/primer3-0.4.0/;Supplementary Table S3). The gene expression of three biological replicates was examined using a MYiQ Single-Color Reverse Transcriptase-Quantitative PCR Detection System (Bio-Rad) using TB Green™ Premix ExTaq™ II (Takara). AcActin (Ciclev10025866m.g) was used as the housekeeping gene after examining its constitutive expression pattern in the RNA-seq results. Relative expression values were calculated using fruit at 196 DAFB (0 d).