Optimization of high quality total RNA isolation from the microalga, Chlorella sp. (Trebouxiophyceae, Chlorophyta) for next‐generation sequencing

Abstract

The advent of high-throughput next-generation sequencing (NGS) has enabled more comprehensive transcriptome analyses to obtain gene expression data and understand metabolic pathways essential for functional analysis and systems biology. The isolation of high purity and intact RNA of sufficient quality and yield is crucial to the success of NGS sequencing. In green microalgae, extraction of nucleic acids is hindered by high concentration of lipids and polysaccharides that co-precipitate with nucleic acids thus resulting in reduced yield and poor quality extracts. The present study compared the performance of standard and modified total RNA isolation protocols using different cell disruption techniques combined with TRIzol reagent or a commercially available kit, in meeting the stringent requirements for downstream NGS application of a green freshwater microalga, Chlorella sp. The protocols were evaluated for (i) the yield of RNA, (ii) integrity of RNA as determined by the RNA integrity number and (iii) the purity of RNA as determined by the A 260 /A 280 and A 260 /A 230 ratios. In general, higher yields were obtained by cell disruption via flash freezing and homogenizing with liquid nitrogen followed by lysis with TRIzol reagent. We recommend the incorporation of a salt precipitation step to improve the purity and integrity of RNA isolated. Results of this study served as a simple and low-cost practical guide for researchers working on isolation of high quality total RNA from microalgae, considering that most relevant publications do not provide a detailed methodology for total RNA isolation or use more expensive methods (e.g. bead-beater).

Determination of Photosynthesis-Related and Ascorbate Peroxidase Gene Expression in the Green Algae (Chlorella vulgaris) Under High-Temperature Conditions

“…Previous studies reported that over gene expressions of APX enhanced the tolerance capacity and minimized photooxidative damage under temperature stress (Caverzan et al, 2012;Sato et al, 2011;Shi et al, 2001;Miller et al, 2007). Park et al (2004) also emphasized the highly induced cAPX gene levels in sweet potato leaves after high-temperature exposure. Moreover, Ma et al (2008) showed the increment expression levels APX in apple leaves at 40˚C for 4 h exposure and decreasing afterward.…”

“…at 15 to 30°C, the samples were centrifuged at 10000 x g at 4°C for 10 min. After washing with 75% ethanol, the pellet was air-dried for 15 min (Poong et al, 2017). The quality and quantity of obtained RNA were measured by spectrophotometer (Pharo 300, Merck).…”

Comparison and improvement of RNA extraction methods in Sargassum (Phaeophyta)

“…In addition, Method 5 has been popularly used in other macroalgae and microalgae, such as Amphiroa fragilissima and Chlorella sp. (Li et al, 2021; Poong et al, 2017; Yang et al, 2021). In this study, we found that it was not appropriate for the three Sargassum species, and this was confirmed by the extremely lower ratios of A260/280 and A260/230, such as 0.19 of 260/230, in S. hemiphyllum and the severe degradation in gel electrophoresis (Table 3 and Figure 2).…”

RNA isolation from taxonomically diverse photosynthetic protists

“…Extraction buffers containing high salt concentrations have been commonly used to separate RNA from polysaccharide‐rich samples (e.g., from Chlorella sp. [Poong et al ]), but also remove small RNAs and therefore put a selection on the extracted nucleic acids (Carra et al ). Furthermore, even small amounts of lithium ions may interfere with downstream applications (Bilgin et al ).…”

“…These commercial protocols are in most cases not applicable for samples containing high levels of phenols, proteins, polysaccharides, or for environmental samples (Tveit et al ). Several of the proposed standard protocols have been modified for the extraction of RNA from macro‐ and microalgae (Chan et al ; Heinrich et al ; Greco et al ; Lopez et al ; Poong et al ), but most of them have only been tested for single species or organisms from similar groups. Taking a closer look, there are only few different buffer systems in use for the extraction of RNA from photosynthetic protists.…”