Simple and rapid RNA extraction from freeze-dried tissue of brown algae and seagrasses

Abstract

An inexpensive and rapid RNA extraction protocol for brown algae and seagrasses is presented, based on homogenization in a simple CTAB buffer and selective precipitation of RNA with lithium chloride. The protocol avoids the use of toxic chaotropic agents and phenol; high concentrations of dithiothreitol are used to inhibit RNase activity and to prevent oxidative cross-linking of nucleic acids by phenolics. A relatively high throughput of about 100 samples in 24 h can be achieved for, for example, Northern analysis. Yields of 50-200 mg g À1 fresh weight are comparable with those obtained for higher plants using commercially available kits. Tests of the extraction procedure demonstrate that high quality, intact RNA can be obtained from a variety of lyophilized brown algal tissues, even after prolonged storage at room temperature. Lyophilization is therefore suggested as an alternative to freezing tissue at À70 C to À80 C. The RNA obtained was used directly in several downstream applications to detect Fucus plastid-encoded transcripts by RNA-labelling, RT-PCR and Northern analysis.

Functional divergence in heat shock response following rapid speciation of Fucus spp. in the Baltic Sea

“…Total RNA was extracted from lyophilized samples following Pearson et al (2006). RNA samples were treated with DNase I (Qiagen, Hilden, Germany) cleaned using RNeasy (Qiagen, Hilden, Germany), quantified by UV spectrophotometry, checked for integrity by agarose gel electrophoresis, and stored at -80°C.…”

Herbivore-induced defence response in the brown seaweedFucus vesiculosus(Phaeophyceae): temporal pattern and gene expression

“…The mixture was incubated at 45°C for 15 min. Afterwards two consecutive chloroform extractions were performed as described by Pearson et al (2006). Subsequently, total RNA was extracted using the Qiagen Plant Mini Kit (Qiagen, Hildesheim, Germany) according to the manufacturer's protocol for RNA extraction, including on-column DNA digestion to ensure as little contamination with DNA as possible due to sensitivity of downstream applications.…”

High-Quality RNA Preparation from Rhodosporidium toruloides and cDNA Library Construction Therewith

“…RNAiso reagent could degrade the cells, dissolve the cell inclusion, keep the integrity of RNA, and probably reduce the contamination of polyphenols and secondary metabolites. b-mercaptoethanol was added to inhibit the RNase and prevent oxidative cross-linking of nucleic acids by polyphenols [40]. Ethanol, sodium acetate, and potassium acetate were used simultaneously to precipitate polysaccharides and remove the lipids [41].…”