ISOLATION OF HIGH QUALITY TOTAL RNA FROM VARIOUS TISSUES OF OIL PALM (Elaeis guineensis) FOR REVERSE TRANSCRIPTION QUANTITATIVE REAL-TIME PCR (RT-qPCR)

Abstract

The extraction of high quality total ribonucleic acid (RNA) has become an essential step for downstream plant molecular biology research. In oil palm, several total RNA extraction protocols have been reported across specific tissues using either conventional method or commercial kits. To our knowledge, there is no specific total RNA extraction method described for oil palm whole fruit which contains lipids, polysaccharides, protein, polyphenols and other secondary metabolites. Here, a modified cetyltrimethylammonium bromidelithium chloride (CTAB-LiCl) method with addition of phenol was established to extract total RNA across various oil palm tissues, especially for whole fruit at eight weeks after anthesis (WAA) and 15 WAA. To assess the extracted total RNA, quality and quantity as well as integrity were evaluated using spectrophotometer and bioanalyser, respectively. It was found that modified CTAB-LiCl method with addition of phenol was able to produce good quality (both A 260 /A 280 and A 260 /A 230 ratios > 1.8) and intact (RIN > 7.4) total RNA. Furthermore, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was successfully performed across all total RNA extracted using two reference genes (PD00569 and pOP-EA01332) and one gene of interest, ethylene-responsive transcription factor-3 like, designated as PD00088. The results indicated that the method is suitable for extraction of total RNA from whole fruit as well as other oil palm tissues and is amenable to RT-qPCR.

Expression of fatty acid and triacylglycerol synthesis genes in interspecific hybrids of oil palm

“…Total RNA was extracted from the mesocarp tissue using the cetyltrimethylammonium bromide method in combination with the silica column of RNeasy Plant Mini Kit (QIA-GEN, Germany), as previously reported for Jatropha curcas L 30 . The method was optimized and modified for oil palm fruit tissues by Ong et al 31 . The total RNA was purified using the RNeasy Mini kit and RNase-free DNase I following the manufacturer's instructions (QIAGEN, Germany).…”