A Simple and Effective Method for High Quality Co-Extraction of Genomic DNA and Total RNA from Low Biomass Ectocarpus siliculosus, the Model Brown Alga

Abstract

The brown seaweed Ectocarpus siliculosus is an emerging model species distributed worldwide in temperate coastal ecosystems. Over 1500 strains of E. siliculosus are available in culture from a broad range of geographic locations and ecological niches. To elucidate the molecular mechanisms underlying its capacity to cope with different environmental and biotic stressors, genomic and transcriptomic studies are necessary; this requires the co-isolation of genomic DNA and total RNA. In brown algae, extraction of nucleic acids is hindered by high concentrations of secondary metabolites that co-precipitate with nucleic acids. Here, we propose a reliable, rapid and cost-effective procedure for the co-isolation of high-quality nucleic acids using small quantities of biomass (25-, 50- and 100 mg) from strains of E. siliculosus (RHO12; LIA4A; EC524 and REP10–11) isolated from sites with different environmental conditions. The procedure employs a high pH extraction buffer (pH 9.5) which contains 100 mM Tris-HCl and 150 mM NaCl, with the addition of 5 mM DTT and 1% sarkosyl to ensure maximum solubility of nucleic acids, effective inhibition of nuclease activity and removal of interfering contaminants (e.g. polysaccharides, polyphenols). The use of sodium acetate together with isopropanol shortened precipitation time and enhanced the yields of DNA/RNA. A phenol:chlorophorm:isoamyl alcohol step was subsequently used to purify the nucleic acids. The present protocol produces high yields of nucleic acids from only 25 mg of fresh algal biomass (0.195 and 0.284 µg mg−1 fresh weigh of RNA and DNA, respectively) and the high quality of the extracted nucleic acids was confirmed through spectrophotometric and electrophoretic analyses. The isolated RNA can be used directly in downstream applications such as RT-PCR and the genomic DNA was suitable for PCR, producing reliable restriction enzyme digestion patterns. Co-isolation of DNA/RNA from different strains indicates that this method is likely to have wider applications for intra- and inter-specific studies on other brown algae.

Reference genes for transcript quantification in Gracilaria tenuistipitata under drought stress

“…Colored squares represent the dehydrated samples a TRX (green), b OEE1 (orange), and c NIA2 (blue) of pure RNA and was quick and efficient, allowing multiple rounds of extractions in a single day (Table 3). The previously described methods, such as the TSS method (Greco 2014) and the protocol described in Falcão (2008), were not as suitable, as their long incubations required a full working day for a single round of extractions. RNeasy had comparatively lower yields to the modified TRIzol method, due to the limited RNA binding capacity of the spin columns and possible clogging by agar but, due to its simplicity, could be attractive if only small quantities of RNA are required.…”

“…The following RNA extraction methods were tested: TRIzol Plus RNA Purification System (Thermo Fisher), PureLink Plant RNA Reagent (Thermo Fisher), RNeasy Plant Mini Kit (QIAGEN), and the Tris-Sarkosyl-saline (TSS) method suggested by Greco et al (2014). The TRIzol and PureLink methods followed the manufacturer's instructions for plant RNA extraction with the following changes: there was an extra precooled absolute ethanol wash after the RNA precipitation, two 5-min incubations to dry the pellet (one with the tube bottom up and the other with the tube bottom down), and a 5-min incubation at 60°C for pellet resuspension.…”

“…Four different protocols were tested considering yield, time required, complexity, and cost: TRIzol Plus RNA Purification System (Thermo Fisher), PureLink Plant RNA Reagent (Thermo Fisher), RNeasy Plant Mini Kit (QIAGEN), and the Tris-Sarkosyl-Saline protocol (TSS) published by Greco et al (2014) for brown algae. TRIzol is a commercial variation of the acid phenol/chloroform guanidinium isothiocyanate RNA extraction (Chomczynski and Sacchi 1987) that facilitates DNA precipitation by acidic pH conditions in extraction solutions.…”

Enzymatic antioxidant defences are transcriptionally regulated in Es524, a copper-tolerant strain of Ectocarpus siliculosus (Ectocarpales, Phaeophyceae)

“…Prior to gene expression analyses on E. siliculosus strain Es524, total RNA was extracted according to our protocol (Greco et al 2014). Specific oligonucleotide primers were designed as described in Roncarati et al (2015).…”

DNA-Based Identification and Chemical Characteristics of Hypnea musciformis from Coastal Sites in Ghana

“…DNA-based identification methods require successful DNA extraction and amplification, which has been widely reported to be difficult for seaweed biomass, due to co-isolation of hydrocolloids and polyphenols [21]. Phenolic compounds bind firmly to DNA during DNA extraction and interfere with subsequent reactions, including during PCR amplification.…”