During a harmful algal bloom (HAB), the seawater contains a high abundance of microorganisms and elemental ions. Such components can interfere with RNA isolation, leading to RNA degradation. The complex HAB seawater property makes isolating high-quality RNA for metatranscriptomic sequencing difficult, which is required for effective RNA sequencing and transcriptome profiling. This study used three isolation techniques to find the optimal strategy for isolating total RNA from bloom samples. One of the isolation techniques was the phenol-chloroform extraction method, which uses organic solvents to isolate RNA. The remaining two isolation techniques used the same commercial RNA extraction kit, TransZol Up Plus RNA kit (TransGen Biotech, China). One followed the extraction kit’s protocol, while the other modified the protocol. Total RNA was extracted from three seawater samples of three occasions of HAB in Sepanggar Bay. The most effective approach used to extract high-quality RNA from the environmental samples of the HABs was the TransZol Up Plus RNA kit, with modified protocol. Results of the modified protocol generated a high-purity total RNA, ranging from 2.081 to 2.474 for both the absorbance ratios A260/280 and A260/230. The RNA integrity number value ranged from 6.2 to 7.6. All of the samples resulted in concentrations up to 91 ng/µl. We concluded that the modified protocol of TransZol Up Plus RNA kit yielded the highest quality total RNA for metatranscriptome next-generation sequencing (NGS). Apart from NGS, the high-quality RNA can also be used for various downstream applications, including real-time PCR, RNA cloning, and RNA microarray analysis.

• Anderson, D. M., Gilbert, P. M., & Burkholder, J. M. (2002). Harmful algal blooms and eutrophication: Nutrient sources, composition, and consequences. Estuaries, 25, 704-726. https://doi.org/10.1007/BF02804901

• Anton, A., Alexander, J., & Estim, A. (2000, February 7-11). Harmful algal blooms in Malaysia: Revisiting Kimanis Bay. In 9th International conference on toxic Phytoplankton (pp. 7-11). Tasmania, Australia.

• Anton, A., Teoh, P. L., Mohd-Shaleh, S. R., & Mohammad-Noor, N. (2008). First occurrence of Cochlodinium blooms in Sabah Malaysia. Harmful Algae, 7(3), 331-336. http://doi.org/10.1016/j.hal.2007.12.013

• Barsanti, L., & Gualtieri, P. (2014). Algae: anatomy, biochemistry, and biotechnology. CRC Press. https://doi.org/10.1201/b16544

• Becker, C., Hammerle-Fickinger, A., Riedmaier, I., & Pfaffl, M. W. (2010). mRNA and microRNA quality control for RT-qPCR analysis. Methods, 50(4), 237-243. https://doi.org/10.1016/j.ymeth.2010.01.010

• Buchmueller, K. L., & Weeks, K. M. (2003). Near native structure in an RNA collapsed state. Biochemistry, 42(47), 13869-13878. https://doi.org/10.1021/bi035476k

• Chin, G. J. W. L., Teoh, P. L., Kumar, S. V., & Anton, A. (2013). Ribosomal DNA analysis of marine microbes associated with toxin-producing Pyrodinium bahamense var. compressum (Bohm), a harmful algal bloom species. Pertanika Journal of Tropical Agricultural Science, 36(2), 179-188.

• 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, 581-585. https://doi.org/10.1038/nprot.2006.83

• Cooper, E. D., Bentlage, B., Gibbons, T. R., Bachvaroff, T. R., & Delwiche, C. F. (2014). Metatranscriptome profiling of a harmful algal bloom. Harmful Algae, 37, 75-83. https://doi.org/10.1016/j.hal.2014.04.016

• Dehghani, M., Rosenblatt, K. P., Li, L., Rakhade, M., & Amato, R. J. (2019). Validation and clinical applications of a comprehensive next generation sequencing system for molecular characterization of solid cancer tissues. Frontiers in Molecular Biosciences, 6, 82. https://doi.org/10.3389/fmolb.2019.00082

• Die, J. V., & Roman, B. (2012). RNA quality assessment: A view from plant qPCR studies. Journal of Experimental Botany, 63(17), 6069-6077. https://doi.org/10.1093/jxb/ers276

• Gallagher, J. B., Hoe, C. C., Yusob, M. S. B. M., Gen, C. N., & Mae, G. Y. (2016). Surface chlorophyll patchiness across Sepanggar Bay: Relationships with turbidity and depth. Transactions on Science and Technology, 3(2), 421-426.

• Goh, C. S., & Lee, K. T. (2010). A visionary and conceptual macroalgae-based third-generation bioethanol (TGB) biorefinery in Sabah, Malaysia as an underlay for renewable and sustainable development. Renewable and Sustainable Energy Reviews, 14(2), 842-848. https://doi.org/10.1016/j.rser.2009.10.001

• Gong, W., Browne, J., Hall, N., Schruth, D., Paerl, H., & Marchetti, A. (2017). Molecular insights into a dinoflagellate bloom. The ISME Journal, 11, 439-452. https://doi.org/10.1038/ismej.2016.129

• Green, M. R., & Sambrook, J. (2020). Precipitation of RNA with ethanol. Cold Spring Harbor Protocols, 89-91. https://doi.org/10.1101/pdb.prot101717

• Harun, S. N., Mohammad-Noor, N., Ahmad, Z., Chu, K. B., Saad, S., Mohamad Hidayat, N. S., & MuKai, Y. (2015). First report of Cochlodinium polykrikoides (Dinophyceae), a harmful algal bloom (HAB) species in the coastal waters of peninsular Malaysia. Malaysian Journal of Science, 34(1), 87-92. https://doi.org/10.22452/mjs.vol34no1.9

• He, J., & Jiao, Y. (2014) Next-generation sequencing applied to flower development: RNA-Seq. In J. Riechmann & F. Wellmer (Eds.), Flower Development: Methods and protocols (pp. 401-411). Humana Press. https://doi.org/10.1007/978-1-4614-9408-9_23

• Islam, M. S., Aryasomayajula, A., & Selvaganapathy, P. R. (2017). A review on macroscale and microscale cell lysis methods. Micromachones, 8(3), 83. https://doi.org/10.3390/mi8030083

• Jeffries, S., Kiss, M. K., Smith, A. J., & Smith, A. W. (2014). A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples. BMC Biotechnology, 14, Article 94. https://doi.org/10.1186/s12896-014-0094-8

• Jiang, L., Schlesinger, F., Davis, C. A., Zhang, Y., Li, R., Salit, M., Gingeras, T. R., & Oliver, B. (2011). Synthetic spike-in standards for RNA-seq experiments. Genome Research, 21, 1543-1551. https://doi.org/10.1101/gr.121095.111

• Jipanin, S. J., Muhamad-Shaleh, S. R., Lim, P. T., Leaw, C. P., & Mustapha, S. (2019). The monitoring of harmful algae blooms in Sabah, Malaysia. Journal of Physics: Conference Series, 1358, Article 012014. https://doi.org/10.1088/1742-6596/1358/1/012014

• Johnson, M. (2012). RNA extraction. Mater Methods, 2, 201. https://doi.org/10.13070/mm.en.2.201

• Karp, A., Isaac, P. G., & Ingram, D. S. (1998). Isolation of nucleic acids using silica-gel based membranes: Methods based on the use of QIAamp Spin Columns. In A. Karp, P. G. Isaac & D. S. Ingram (Eds.), Molecular Tools for Screening Biodiversity (pp. 59-63). Springer. https://doi.org/10.1007/978-94-009-0019-6_14

• Kaser, M., Ruf, M. T., Hauser, J., Marsollier, L., & Pluschke, G. (2009). Optimized method for preparation of DNA from pathogenic and environmental mycobacteria. Applied and Environmental Microbiology, 75(2), 414-418. https://doi.org/10.1128/AEM.01358-08

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

• Law, S. V., Rodrigues, K. F., Jani, J., Anton, A., & Chin, G. J. W. L. (2020). A metagenomic study of bacterial communities associated with the saxitoxin producing dinoflagellate, Pyrodinium bahamense var. compressum. Malaysian Journal of Microbiology, 16(3),176-183. https://doi.org/10.21161/mjm.190548

• Leaw, C. P., Lim, P. T., Ng, B. K., Cheah, M. Y., Ahmad, A., & Usup, G. (2005). Phylogenetic analysis of Alexandrium species and Pyrodinium bahamense (Dinophyceae) based on theca morphology and nuclear ribosomal gene sequence. Phycologia, 44(5), 550-565.

• Liaw, J., & Fung, S., (2000). Sustainable marine aquaculture in Sabah. Berita IDS, 15, 1-2.

• Lin, X., Mckinley, J., Resch, C. T., Kaluzny, R., Lauber, R. L., Fredrickson, J., Knight, R., & Konopka, A. (2012). Spatial and temporal dynamics of the microbial community in the Hanford unconfined aquifer. The ISME Journal, 6, 1665-1676. https://doi.org/10.1038/ismej.2012.26

• Lucena-Aguilar, G., Sanchez-Lopez, A. M., Barberan-Aceituno, C., Carrillo-Avi;a, J. A., Lopez-Guerrero, J. A., & Aguilar-Quesada, R. (2016). DNA source selection for downstream applications based on DNA quality indicators analysis. Biopreservation and Biobanking, 14(4), 264-270. https://doi.org/10.1089/bio.2015.0064

• Mäki, A., Salmi, P., Mikkonen, A., Kremp A., & Tiirola, M. (2017). Sample preservation, DNA or RNA extraction and data analysis for high-throughput phytoplankton community sequencing. Frontiers in Microbiology, 8, Article 1848. https://doi.org/10.3389/fmicb.2017.01848

• Mohammad-Noor, N., Adam, A., Franco Soler, J. M., Anton, A., & Sitti-Raehanah, M. S. (2010). First record of toxic Gymnodinium catenatum off the west coast of Sabah, Malaysia. In K. C. Ho, M. J. Zhou & Y. Z. Qi (Eds.), Proceedings of 13th International Conference on Harmful Algae (p. 25). International Society for the Study of Harmful Algae.

• Mutz, K. O., Heilkenbrinker, A., Lonne, M., Walter, J. L., & Stahl, F. (2013). Transcriptome analysis using next-generation sequencing. Current Opinion in Biotechnology, 24(1), 22-30. https://doi.org/10.1016/j.copbio.2012.09.004

• Palani-Velu, K. D., Chin, G. J. W. L., Rodrigues, K. F., & Anton, A. (2013). Optimization of total RNA isolation from Cochlodinium polykrikoides. Journal of Applied Phycology, 25, 59-64. https://doi.org/10.1007/s10811-012-9838-9

• Roberts, V. A., Vigar, M., Backer, L., Veytsel, G. E., Hilborn, E. D., Hamelin, E. I., Vanden Esschert, K. L., Lively, J. Y., Cope, J. R., Hlavsa, E. I., & Yoder, J. S. (2020). Surveillance for harmful algal bloom events and associated human and animal illnesses - One health harmful algal bloom system, United States, 2016-2018. Morbidity and Mortality Weekly Report, 69(50), 1889-1894. https://doi.org/10.15585/mmwr.mm6950a2

• Roy, R. N. (1977). Red tide and outbreak of paralytic shellfish poisoning in Sabah. Medical Journal of Malaysia, 31(3), 247-251.

• Sambrook, J., & Russell, D. W. (2006). Purification of nucleic acids by extraction with phenol: chloroform. Cold Spring Harbor Protocols, (1), pdb-prot4455. https://doi.org/10.1101/pdb.prot4455

• Schmid, M. W., Schmidt, A., Klostermeier, U. C., Barann, M., Rosenstiel, P., & Grossniklaus, U. (2012). A powerful method for transcriptional profiling of specific cell types in eukaryotes: Laser-assisted microdissection and RNA sequencing. PLoS One, 7(1), Article e29685. https://doi.org/10.1371/journal.pone.0029685

• 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, Article 3. https://doi.org/10.1186/1471-2199-7-3

• Shin, J. H. (2012). Nucleic acid extraction techniques. In Y. W. Tang & C. Stratton (Eds.), Advanced Techniques in Diagnostic Microbiology (pp. 209-225). Springer. https://doi.org/10.1007/978-1-4614-3970-7_11

• Tan, K. S., & Ransangan, J. (2016). Effects of environmental conditions and nutrients on the occurrence and distribution of potentially harmful phytoplankton in mesotrophic water. Sains Malaysiana, 45(6), 865-877.

• Tan, S. C., & Yiap, B. C. (2009). DNA, RNA, and protein extraction: The past and the present. BioMed Research International, 2009, Article 574398. https://doi.org/10.1155/2009/574398

• Tavares, L., Alves, P. M., Ferreira, R. B., & Santos, C. N. (2011). Comparison of different methods for DNA-free RNA isolation from SK-N-MC neuroblastoma. BMC Research Notes, 4, Article 3. https://doi.org/10.1186/1756-0500-4-3

• Thompson, K. L., Pine, P. S., Rosenzweig, B. A., Turpaz, Y., & Retief, J. (2007). Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA. BMC Biotechnology. 7, Article 57. https://doi.org/10.1186/1472-6750-7-57

• Usup, G., & Azanza, R. V. (1998). Physiology and bloom dynamics of the tropical dinoflagellate, Pyrodinium bahamense. In D. M. Anderson, A D. Cembella & G. M. Hallegraeff (Eds.), Physiological ecology of harmful algal blooms. Springer.

• Usup, G., Ahmad, A., & Ismail, N. (1989). Pyrodinium bahamense var. compressum red tide studies in Sabah, Malaysia. In G. M. Hallegraeff & J. L. Maclean (Eds.), Biology, Epidemiology and Management of Pyrodinium red tides (pp. 97-110). Fisheries Department, Ministry of Development, Philippines.

• Usup, G., Ahmad, A., Matsuoka, K., Lim, P. T., & Leaw, C. P. (2012). Biology, ecology, and bloom dynamics of the toxic marine dinoflagellate Pyrodinium bahamense. Harmful Algae, 14, 301-312. https://doi.org/10.1016/j.hal.2011.10.026

• Wang, Y., Hayatsu, M., & Fujii, T. (2012). Extraction of bacterial RNA from soil: Challenges and solutions. Microbes and Environments, 27(2), 111-121. https://doi.org/10.1264/jsme2.ME11304

• Woodson, S. A., & Koculi, E. (2009). Analysis of RNA folding by native polyacrylamide gel electrophoresis. Methods Enzymology, 469, 189-208. https://doi.org/10.1016/S0076-6879(09)69009-1

• Wu, M., & Tinoco, I. (1998). RNA folding causes secondary structure rearrangement. Proceedings of the National. Academy of Sciences of the United States of America, 95(20), 11555-11560. https://doi.org/10.1073/pnas.95.20.11555

• Xu, S., He, C., Kang, Z., Song, S., & Li, C. (2021). The ecological responses of bacterioplankton during a Phaeocystis globosa bloom in Beibu Gulf, China highlighted by integrated metagenomics and metatranscriptomics. Research Square. https://doi.org/10.21203/rs.3.rs-1011939/v1

• Xu, X., Yu, Z., He, L., Cao, X., Chen, N., & Song, X. (2020). Metabolic analyses by metatranscriptomics highlight plasticity in phosphorus acquisition during monospecific and multispecies algal blooms. Hydrobiologia, 847, 1071-1085. https://doi.org/10.1007/s10750-019-04169-x