PERTANIKA JOURNAL OF TROPICAL AGRICULTURAL SCIENCE

High-quality RNA is an important genetic study as it has minimal contaminants that can affect gene discovery including degraded RNAs, chemical, and biological residues. Hence, it is a prerequisite for genetic analysis using Next Generation Sequencing (NGS) for accurate and reliable data mining. Despite its importance, extracting high-quality RNA from different samples is often a challenge, as every tissue has a different biochemical composition, thus requiring different protocols. This paper reports protocols for the extraction of high-quality RNA from two type of pineapple tissues, which are thickly lignified hard tissue (tillers, inflorescence, flowers) and watery soft tissue (mature fruit, ripe fruit, and overripe fruit) via modified Kim and Hamada (2005) method. Total RNA was extracted in all six tissues, which showed two distinctive 25S and 18S band on agarose gel. The total RNA in this study was considered high-quality as the minimum concentration was 50 ng/μl, the absorbance ratio (A260:A280) was more than 1.8 and RNA integrity number (RIN) was greater than 7. The obtained results showed that the modified Kim and Hamada (2005) method was effective in extracting high-quality RNA from the challenging MD2 pineapple tissue, which is suitable for subsequent molecular analysis, including the highly sensitive NGS.

• Atshan, S. S., Shamsudin, M. N., Lung, L. T., Ling, K. H., Sekawi, Z., Pei, C. P., & Ghaznavi-Rad, E. (2012). Improved method for the isolation of RNA from bacteria refractory to disruption, including S. aureus producing biofilm. Gene, 494(2), 219–224. https://doi.org/10.1016/j.gene.2011.12.010

• Azmat, M. A., Khan, I. A., Cheema, H. M., Rajwana, I. A., Khan, A. S., & Khan, A. A. (2012). Extraction of DNA suitable for PCR applications from mature leaves of Mangifera indica L.. Journal of Zhejiang University Science B, 13(4), 239–243. https://doi.org/10.1631/jzus.B1100194

• Bakar, B. H., Ishak, A. J., Shamsuddin, R., & Hassan, W. Z. (2013). Ripeness level classification for pineapple using RGB and HSI colour maps. Journal of Theoretical and Applied Information Technology, 57(3), 587-593.

• Chaparro-Encinas, L. A., Arellano-Wattenbarger, G. L., Parra-Cota, F. I., & Santos-Villalobos, S. (2020). A modified CTAB and Trizol® protocol for high-quality RNA extraction from whole wheat seedlings, including rhizosphere. Cereal Research Communications, 48(3), 275–282. https://doi.org/10.1007/s42976-020-00046-9

• Chomczynski, P., & Sacchi, N. (2006). The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: Twenty-something years on. Nature Protocols, 1(2), 581–585. https://doi.org/10.1038/nprot.2006.83

• Cordenunsi, B., Saura-Calixto, F., Diaz-Rubio, M. E., Zuleta, A., Tiné, M. A., Buckeridge, M. S., Silva, G. B. D., Carpio, C., Giuntini, E. B., Menezes, E. W. D., & Lajolo, F. (2010). Carbohydrate composition of ripe pineapple (cv. Perola) and the glycemic response in humans. Food Science and Technology, 30(1), 282-288. https://doi.org/10.1590/S0101-20612010000100041

• Cox, R. A. (1968). The use of guanidinium chloride in the isolation of nucleic acids. Methods in Enzymology, 12(Part B), 120–129. https://doi.org/10.1016/0076-6879(67)12123-X

• Ghangal, R., Raghuvanshi, S., & Sharma, P., C. (2009). Isolation of good quality RNA from a medicinal plant seabuckthorn, rich in secondary metabolites. Plant Physiology and Biochemistry, 47(11), 1113–15. https://doi.org/10.1016/j.plaphy.2009.09.004

• Godden, G. T., Jordon-Thaden, I. E., Chamala, S., Crowl, A. A., García, N., Germain-Aubrey, C. C., Heaney, J. M., Latvis, M., Qi, X., & Gitzendanner, M. A. (2012). Making next-generation sequencing work for you: Approaches and practical considerations for marker development and phylogenetics. Plant Ecology and Diversity, 5(4), 427–450. https://doi.org/10.1080/17550874.2012.745909

• Holmes, A., Birse, L., Jackson, R. W., & Holden, N. J. (2014). An optimized method for the extraction of bacterial mRNA from plant roots infected with Escherichia coli O157:H7. Frontiers in Microbiology, 5, 286. https://doi.org/10.3389/fmicb.2014.00286

• Hopkins, J. F., Panja, S., McNeil, S. A. N., & Woodson, S. A., (2009). Effect of salt and RNA structure on annealing and strand displacement by Hfq. Nucleic Acids Research, 37(18), 6205–6213. https://doi.org/10.1093/nar/gkp646

• Hossain, M. F., Akhtar, S., & Anwar, M. (2015). Nutritional value and medicinal benefits of pineapple. International Journal of Nutrition and Food Sciences, 4(1), 84-88. https://doi.org/10.11648/j.ijnfs.20150401.22

• Hou, P., Xie, Z., Zhang, L., Song, Z., Mi, J., He, Y., & Li, Y. (2011) Comparison of three different methods for total RNA extraction from Fritillaria unibracteata: A rare Chinese medicinal plant. Journal of Medicinal Plants Research, 5(13), 2835-2839. https://doi.org/10.5897/JMPR.9000389

• Huded, A., Jingade, P., & Mishra, M. K. (2018). A rapid and efficient SDS-based RNA isolation protocol from different tissues of coffee. 3 Biotech, 8(3), 183. https://doi.org/10.1007/s13205-018-1209-z

• Jaakola, L., Pirttilä, A. M., Halonen, M., & Hohtola, A. (2001). Isolation of high quality RNA from bilberry (Vaccinium myrtillus L.) fruit. Molecular Biotechnology, 19(2), 201–203. https://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. https://doi.org/10.3732/apps.1400105

• Kałużna, M., Kuras, A., Mikiciński, A., & Puławska, J. (2016). Evaluation of different RNA extraction methods for high-quality total RNA and mRNA from Erwinia amylovora in planta. European Journal of Plant Pathology, 146(4), 893–899. https://doi.org/10.1007/s10658-016-0967-x

• Kim, S. H., & Hamada, T. (2005). Rapid and reliable method of extracting DNA and RNA from sweetpotato, Ipomoea batatas (L). Lam. Biotechnology Letters, 27(23), 1841–1845. https://doi.org/10.1007/s10529-005-3891-2

• Kukurba, K. R., & Montgomery, S. B. (2015). RNA sequencing and analysis. Cold Spring Harbor Protocols, 2015(11), 951–969. https://doi.org/10.1101/pdb.top084970

• Li, D., Ren, W., Wang, X., Wang, F., Gao, Y., Ning, Q., Han, Y., Song, T., & Lu, S. (2009). A modified method using TRIzol® reagent and Liquid nitrogen produces high-quality RNA from rat pancreas. Applied Biochemistry and Biotechnology, 158(2), 253-61. https://doi.org/10.1007/s12010-008-8391-0

• Li, X., Wang, C., Sun, H., & Li, T. (2011). Establishment of the total RNA extraction system for lily bulbs with abundant polysaccharides. African Journal of Biotechnology, 10(78), 17907–17915. https://doi.org/10.5897/AJB10.2523

• Liu, L., Han, R., Yu, N., Zhang, W., Xing, L., Xie, D., & Peng, D. (2018). A method for extracting high-quality total RNA from plant rich in polysaccharides and polyphenols using Dendrobium huoshanense. PLOS One, 13(5), 1–9. https://doi.org/10.1371/journal.pone.0196592

• Ma, Z., Huang, B., Xu, S., Chen, Y., Li, S., & Lin, S. (2015) Isolation of high-quality total RNA from Chinese Fir (Cunninghamia lanceolata (Lamb.) Hook). PLOS One, 10(6), e0130234. https://doi.org/10.1371/journal.pone.0130234

• Maes, M., & Messens, E. (1992). Phenol as grinding material in RNA preparations. Nucleic Acids Research, 20(16), 4374. https://doi.org/10.1093/nar/20.16.4374

• Ramimoghadam, D., Hussein, M. Z., & Taufiq-Yap, Y. H. (2012). The effect of sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB) on the properties of ZnO synthesized by hydrothermal method. International Journal of Molecular Science, 13(10), 13275-13293. https://doi.org/10.3390/ijms131013275

• Shamsudin, R., Daud, W., Takriff, M., & Hassan, O. (2007). Physicochemical properties of the Josapine variety of pineapple fruit. International Journal of Food Engineering, 3(5), 14. https://doi.org/10.2202/1556-3758.1115

• Sheng, Q., Vickers, K., Zhao, S., Wang, J., Samuels, D. C., Koues, O., Shyr, Y., & Guo, Y. (2017). Multi-perspective quality control of Illumina RNA sequencing data analysis. Briefings in Functional Genomics, 16(4), 194–204. https://doi.org/10.1093/bfgp/elw035

• Soltis, D. E., Gitzendanner, M. A., Stull, G., Chester, M., Chanderbali, A., Chamala, S., Jordan-Thaden, I., Soltis, P. S., Schnable, P. S., & Barbazuk, W. B. (2013). The potential of genomics in plant systematic. Taxon, 62(5), 886-898. https://doi.org/10.12705/625.13

• Tan, S. C., & Yiap, B. C. (2009). DNA, RNA, and protein extraction: The past and the present. Journal of Biomedicine and Biotechnology, 2009, 574398. https://doi.org/10.1155/2009/574398

• Tel-zur, N., Abbo, S., Myslabodski, D., & Mizrahi, Y. (1999). Modified CTAB procedure for DNA isolation from epiphytic cacti of the genera Hylocereus and Selenicereus (Cactaceae). Plant Molecular Biology Reporter, 17(3), 249–254. https://doi.org/10.1023/A:1007656315275

• Tüzmen, S., Baskin, Y., Nursal, A. F., Eraslan, S., Esemen, Y., Çalibas, G., Demir, A. B., Abbasoglu, D., & Hizel, C. (2018). Techniques for nucleic acid engineering: The foundation of gene manipulation. In D. Barh & V. Azevedo (Eds.), Omics technologies and bio-engineering: Towards improving quality of life (pp. 247–315). Academic Press. https://doi.org/10.1016/B978-0-12-804659-3.00014-2

• Wang, X. X., Wang, B., Liu, L. J., Cui, X. P., Yang, J. Y., Wang, H., Jiang, H., Luo, B. B., Long, Z., Dou, W. X., Zhang, N., & Peng, D. X. (2010). Isolation of high-quality RNA and construction of a suppression subtractive hybridization library from ramie (Boehmeria nivea L. Gaud.). Molecular Biology Reports, 37(4), 2099-2103. https://doi.org/10.1007/s11033-009-9671-7

• White, E. J., Venter, M., Hiten, N. F., & Burger, J. T. (2008). Modified cetyltrimethylammonium bromide method improves robustness and versatility: The benchmark for plant RNA extraction. Biotechnology Journal, 3(11), 1424-1428. https://doi.org/10.1002/biot.200800207

• Wong, L. M., Silvaraj, S., & Phoon, L. Q., (2014). An optimised high-salt CTAB protocol for both DNA and RNA isolation from succulent stems of Hylocereus sp.. Journal of Medical Bioengineering, 3(4), 236-240. https://doi.org/10.12720/jomb.3.4.236-240

• Yahia, E., & Carrillo-López, A. (Eds.) (2018). Postharvest physiology and biochemistry of fruits and vegetables (1st ed.). Woodhead Publishing.

• Yu, D., Tang, H., Zhang, Y., Du, Z., Yu, H., & Chen, Q. (2012). Comparison and improvement of different methods of RNA isolation from strawberry (Fragria × ananassa). Journal of Agricultural Science, 4(7), 51-56. https://doi.org/10.5539/jas.v4n7p51