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 mangostanaL.) untuk Penjujukan)

 

AZHANI ABDUL-RAHMAN, NAJAH IZZATI SULEMAN, WAN ADIBAH ZAKARIA, HOE-HAN, GOH NORMAH MOHD NOOR & WAN MOHD AIZAT*

 

Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

 

Diserahkan: 3 Jun 2016/Diterima: 7 Februari 2017

 

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, modified 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 A260/A280 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 didapati menjadi kaedah terbaik untuk mendapatkan RNA tulen (A260/A280 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

RUJUKAN

Akao, Y., Nakagawa, Y., Iinuma, M. & Nozawa, Y. 2008. Anti-cancer effects of xanthones from pericarps of mangosteen. International Journal of Molecular Sciences 9(3): 355-370.

Chan, K.L., Ho, C.L., Namasivayam, P. & Napis, S. 2007. A simple and rapid method for RNA isolation from plant tissues with high phenolic compounds and polysaccharides. Protocol Exchange. doi.org/10.1038/nprot.2007.184.

Dautt-Castro, M., Ochoa-Leyva, A., Contreras-Vergara, C.A., Pacheco-Sanchez, M.A., Casas-Flores, S., Sanchez- Flores, A., Kuhn, D.N. & Islas-Osuna, M.A. 2015. Mango (Mangifera indicaL.) cv. Kent fruit mesocarp de novo transcriptome assembly identifies gene families important for ripening. Frontiers in Plant Science 6: 62. doi.org/10.3389/ fpls.2015.00062.

Gambino, G., Perrone, I. & Gribaudo, I. 2008. A rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants. Phytochemical Analysis 19(6): 520-525. doi.org/10.1002/pca.1078.

Garcia-Seco, D., Zhang, Y., Gutierrez-Mañero, F.J., Martin, C. & Ramos-Solano, B. 2015. RNA-Seq analysis and transcriptome assembly for blackberry (Rubus sp. var. lochness) fruit. BMC Genomics 16(1): 5. doi.org/10.1186/s12864-014-1198-1.

Guo, S., Sun, H., Zhang, H., Liu, J., Ren, Y., Gong, G., Jiao, C., Zheng, Y., Yang, W., Fei, Z. & Xu, Y. 2015. Comparative transcriptome analysis of cultivated and wild watermelon during fruit development. Plos One 10(6): e0130267. doi. org/10.1371/journal.pone.0130267.

Jaakola, L., Pirttilä, A.M., Halonen, M. & Hohtola, A. 2001. Isolation of high quality RNA from bilberry (Vaccinium myrtillusL.) fruit. Molecular Biotechnology 19(2): 201-203. doi.org/10.1385/MB:19:2:201.

Jordon-Thaden, I.E., Chanderbali, A.S., Gitzendanner, M.A. & Soltis, D.E. 2015. Modified CTAB and TRIzol protocols improve RNA extraction from chemically complex embryophyta. Applications in Plant Sciences 3(5): 1400105. doi.org/10.3732/apps.1400105.

Kim, S. & Hamada, T. 2005. Rapid and reliable method of extracting DNA and RNA from sweet potato, Ipomoea batatas(L). lam. Biotechnology Letters 27(23-24): 1841- 1845. doi.org/10.1007/s10529-005-3891-2.

Loomis, W.D. 1974. Overcoming problems of phenolics and quinones in the isolation of plant enzymes and organelles. Methods in Enzymology 31(C): 528-544. doi. org/10.1016/0076-6879(74)31057-9.

López-Gómez, R. & Gómez-Lim, M.A. 1992. A method for extracting intact RNA from fruits rich in polysaccharides using ripe mango mesocarp. HortScience 27(5): 440-442.

Morozova, O. & Marra, M.A. 2008. Applications of next-generation sequencing technologies in functional genomics. Genomics 92(5): 255-264. doi.org/10.1016/j. ygeno.2008.07.001.

Osman, B.M. & Milan, A.R. 2006. Mangosteen: Garcinia mangostana L. Postharvest Biology and Technology of Tropical and Subtropical Fruits: Volume 4: Mangosteen to White Sapote. doi.org/10.1533/9780857092618.1.

Palapol, Y., Ketsa, S., Kui, L-W., Ferguson, I.B. & Allan, A.C. 2009. A MYB transcription factor regulates anthocyanin biosynthesis in mangosteen (Garcinia mangostana L.) fruit during ripening. Planta 229: 1323-1334. doi.org/10.1007/s00425-009-0917-3

Pedraza-Chaverri, J., Cárdenas-Rodríguez, N., Orozco-Ibarra, M. & Pérez-Rojas, J.M. 2008. Medicinal properties of mangosteen (Garcinia mangostana). Food and Chemical Toxicology 46(10): 3227-3239. doi.org/10.1016/j.fct.2008.07.024.

Schroeder, A., Mueller, O., Stocker, S., Salowsky, R., Leiber, M., Gassmann, M., Lightfoot, S., Menzel, W., Granzow, M. & Ragg, T. 2006. The RIN: An RNA integrity number for assigning integrity values to RNA measurements. BMC Molecular Biology 7(1): 3. doi.org/10.1186/1471-2199-7-3.

Shan, T., Ma, Q., Guo, K., Liu, J., Li, W., Wang, F. & Wu, E. 2011. Xanthones from mangosteen extracts as natural chemopreventive agents: Potential anticancer drugs. Current Molecular Medicine 11(8): 666-677. doi. org/10.2174/156652411797536679.

Shu, C., Sun, S., Chen, J., Chen, J. & Zhou, E. 2014. Comparison of different methods for total RNA extraction from sclerotia of Rhizoctonia solani. Electronic Journal of Biotechnology 17(1): 50-54. doi.org/10.1016/j.ejbt.2013.12.009.

Soon, H-K., Yoo, K-L., Ju, B-H. & In, G-L. 1997. A simple method for RNA extraction from marine macroalgae. Algae 12(1): 53-56.

Vasanthaiah, H.K.N., Katam, R. & Sheikh, M.B. 2008. Efficient protocol for isolation of functional RNA from different grape tissue rich in polyphenols and polysaccharides for gene expression studies. Electronic Journal of Biotechnology 11(3): 1-8. doi.org/10.2225/vol11-issue3-fulltext-5.

Wang, Z., Gerstein, M. & Snyder, M. 2009. RNA-Seq: A revolutionary tool for transcriptomics. Nature Reviews Genetics 10(1): 57-63. doi.org/10.1038/nrg2484.

Yockteng, R., Almeida, A.M.R., Yee, S., Andre, T., Hill, C. & Specht, C.D. 2013. A method for extracting high-quality RNA from diverse plants for next-generation sequencing and gene expression analyses. Applications in Plant Sciences 1(12): 1300070. doi.org/10.3732/apps.1300070.

 

 

*Pengarang untuk surat-menyurat; email: wma@ukm.edu.my

 

 

 

 

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