Sains Malaysiana 46(8)(2017): 1231–1240 http://dx.doi.org/10.17576/jsm-2017-4608-08 RNA Extractions of Mangosteen (Garcinia mangostana L.) Pericarps for Sequencing (Pengekstrakan RNA Perikarpa Manggis (Garcinia mangostana L.) untuk Penjujukan) AZHANI ABDUL-RAHMAN, NAJAH IZZATI SULEMAN, WAN ADIBAH ZAKARIA, HOE-HAN GOH, NORMAH MOHD NOOR & WAN MOHD AIZAT* ABSTRACT This study employed several RNA extraction methods for mangosteen pericarps prior to RNA sequencing. The sequencing platform heavily relies on a high quality RNA yield. However, pericarp tissues contain a lot of phenolic compounds that results in low RNA quality. Hence, we studied several RNA extraction methods to obtain the most suitable method for the best RNA quality from the pericarps of mangosteen. Five different methods including Lopez and Gomez, modifed hexadecyltrimethyl ammonium bromide (CTAB) method, several commercial kits from TranszolUP, Favorgen and Qiagen RNeasy were compared. By optimising the CTAB method, it was found to be the best method to obtain pure RNA (high A 260 / A 280 ratio) with the highest yields (up to approximately 600-800 ng/µL concentration). The QC control of these samples using bioanalyzer validated their suitability for the downstream RNA sequencing. This report details the method for extracting high quality and high yield RNA samples from fruit that are rich in polyphenolic compounds such as mangosteen. Keywords: Mangosteen; pericarp; RNA extractions; RNA-seq ABSTRAK Platform penjujukan bergantung sepenuhnya kepada hasil RNA yang berkualiti tinggi. Walau bagaimanapun, tisu kulit manggis mengandungi banyak sebatian fenol yang menyebabkan kualiti RNA menjadi rendah. Oleh itu, penyelidikan ini mengkaji beberapa kaedah pengekstrakan RNA untuk mendapatkan kaedah yang paling sesuai bagi penghasilan RNA yang berkualiti tinggi daripada perikarpa manggis. Lima kaedah yang berbeza termasuk kaedah Lopez dan Gomez, kaedah yang diubah suai daripada heksadesiltrimetil ammonium bromida (CTAB), beberapa kit komersial daripada TranszolUP, Favorgen dan Qiagen Rneasy telah dibandingkan. Dengan mengoptimumkan kaedah CTAB, ia telah didapati menjadi kaedah terbaik untuk mendapatkan RNA tulen (A 260 /A 280 nisbah tinggi) dengan kadar hasil tertinggi (kepekatan sehingga kira-kira 600-800 ng/µL). Kawalan QC sampel ini menggunakan bioanalisis untuk disahkan kesesuaiannya bagi analisis hiliran penjujukan RNA. Laporan ini memperincikan kaedah untuk mengekstrak hasil RNA yang banyak dan berkualiti tinggi daripada buah-buahan yang kaya dengan sebatian polifenol seperti manggis. Kata kunci: Manggis; pengekstrakan RNA; perikarpa; RNA-seq INTRODUCTION Mangosteen ( Garcinia mangostana L.) is a tropical climacteric fruit from the family of Clusiaceae (Guttiferae) cultivated in Southeast Asia countries such as Malaysia, Thailand, Indonesia and Philippines (Pedraza-Chaverri et al. 2008). Apart from its sweet pulp with unique taste, mangosteen is also attractive due to natural phenolic compounds such as xanthones and flavonoids that mostly reside in its purple-coloured pericarp (Figure 1). These bioactive compounds are known to have medicinal properties such as anticarcinogenic, anti- infammatory and antioxidant (Akao et al. 2008; Shan et al. 2011). Interestingly, the molecular mechanism of these compounds in mangosteen has not been elucidated and systems biology approach such as transcriptomics can be utilised to catalogue its gene expression. However, RNA extraction for the transcriptomics approach from mangosteen pericarp samples posed several signifcant problems. The phenolic compounds and polysaccharides are known to co-precipitate and interact irreversibly with nucleic acids which complicate the downstream molecular analysis such as RNA sequencing (Loomis 1974). Furthermore, the thick and hard layer of the pericarp as well as the presence of latex contributes to the diffculties in handling mangosteen samples for RNA extraction. Moreover, the abundance of anthocyanin can also co- precipitate with nucleic acids which resulted in a coloured RNA pellet, jeopardising its purity. The diffculties of RNA extraction from plant tissues that are rich in polysaccharides such as grapevine, mango and sweet potatoes have been reported in many previous studies (Gambino et al. 2008; Kim & Hamada 2005; López-Gómez & Gómez-Lim 1992). Different conditions/ parameters are therefore required to isolate a good quality RNA for different species, different tissues as well as for the same species but grown under different environments