Polyvinylpyrrolidone pvp 40

This protocol was done according to Doyle and Doyle [31 ] with some modification reported by Masoomi-Aladizgeh et al. [28 (link)]. Briefly, CTAB buffer was prepared by dissolving 0.5 g cetyltrimethylammonium bromide (CTAB) (Biochemica, UK), 1 g EDTA disodium dihydrate (Duchefa Biochemie, NLD), 2.5 g Tris base (Sigma, USA) and 5 g NaCl (Duchefa Biochemie, NLD) in 100 mL autoclaved double distilled water on shaker at room temperature. Next, 15 mg/mL polyvinylpyrrolidone (PVP) 40 (Sigma-Aldrich, USA) and 10 µL/mL β-mercaptoethanol 98% (Sigma, USA) were added to the solution and the CTAB buffer was incubated (60 °C for 20 min). For RNA extraction, 1 mL CTAB buffer and 20 µL β-mercaptoethanol were added to 200 mg fine ground sample, mixed well and kept in bain-marie for 10 min at 60 °C. Then, 600 µL chloroform:Isoamyl alcohol (IAA) (Merck, USA) (24:1) was added to each sample and centrifuged (10 min, 10,000 rpm, 4 °C). Subsequently, 700 µL isopropanol (− 20 °C) (Merck, USA) was added to the supernatant and centrifuged again. Pellet was washed with 1 mL ice cold 96% (v/v) ethanol (− 20 °C) and centrifuged for 2 min at 7500 rpm and 4 °C then was dried in sterile air under chemical hood. Finally, the pellet was resuspended in 45 µL DEPC treated deionized water.