RNA Isolation from Environmental Samples of a Harmful Algal Bloom for Metatranscriptome Next-Generation Sequencing

Abstract

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.