2.2. Oil Palm Mesocarp RNA Extraction
Total RNA was extracted from the oil palm mesocarp tissue using the following RNA extraction method. The mesocarp tissue was first ground to a fine powder in liquid nitrogen. The extraction buffer contained 0.1 M Tris-HCl, pH 7.6, 0.1 M NaCl, 6% p-aminosalicylic acid, 1% SDS, and 0.35% β-mercaptoethanol. The buffer was added to frozen ground material at the rate of 4 mL/g plant tissue, vortexed thorough and extracted with phenol/chloroform. An additional chloroform:isoamylalcohol cleanup step was performed after the phenol/chloroform extraction steps to improve the purity of the RNA. The supernatant was precipitated using 3 M LiCl followed by an ethanol precipitation. The pellet was redissolved in DEPC-treated distilled water and again precipitated with 3 M LiCl followed by another round of ethanol precipitation. The precipitate was finally dissolved in DEPC treated distilled water and stored at −70 °C.
The concentration and purity of total RNA was determined by spectrophotometric quantification (Thermo Scientific, Nanodrop ND-1000, Wilmington, DE, USA). The AU 260/280 and AU 260/230 were measured and samples with a ratio of 1.8–2.0 were utilized. Gel electrophoresis was also performed on 1 µg of total RNA using a 1% agarose gel in TAE buffer to further determine the integrity and quality of the RNA. Samples that passed the initial quality tests were further analyzed using an Agilent Bioanalyzer (Agilent, Bioanalyzer 2100, Santa Clara, CA, USA), allowing the ratio of the 28S to 18S peaks to be determined. Samples with 28S to 18S ratios of greater than 2:1 and an RIN (RNA Integrity Number) score greater than 7 (out of 10) were utilized for further work.