Figure 1.
Summary of nucleic acids extraction from E. siliculosus brown alga.
High yields of good quality DNA and RNA are isolated from as little as 25Steps 1–5: Harvested tissue is immediately homogenised using commercial 3 mm solid-glass beads in the presence of 1 mL EB containing 100 mM Tris-HCl, 150 mM NaCl, 5 mM DTT and 1% sarkosyl. These stages allow the lysis of the cell wall, the release of highest amount of nucleic acids, the inactivation of cellular nucleases, and the removal of most of the polysaccharides and other insoluble material. Steps 6–10: Simultaneous presence of absolute ethanol and potassium acetate aids polysaccharide precipitation. Moreover proteins, lipids, pigments and cell debris are removed through extraction of the aqueous phase with chloroform. Steps 11–12: Nucleic acids are then recovered by precipitation with 0.8 V of isopropanol and 0.1 V of 3 M sodium acetate (pH 5.2) in the presence of 1% 2-mercaptoetanol at −80°C. During the precipitation step, salts and other solutes are separated from nucleic acids that form a white precipitate collected by centrifugation. The excess of isopropanol and 2-mercaptoetanol are removed through washing the pellet with 75% ethanol. Step 13: All traces of ethanol are removed, the nucleic acid pellet is dried and resuspended in nuclease-free water. After RNase or DNase treatment the superfluous quantities of proteins, polysaccharides, lipids, and cell debris were removed from the extracted DNA and RNA through double extended purification treatment with phenol:chloroform:isoamyl alcohol.
Table 1.
Split and precipitate the aqueous phase of one sample in more tubes (usually two).
Table 2.
Reagent used in the precipitation step.
Table 3.
The nucleic acid of one sample precipitated in two different tubes is transferred in one tube after the resuspension in the appropriate volume of nuclease-free water.
Table 4.
Comparison of pure DNA yield and purity, obtained from four strains of E. siliculosus by two different methods: the new and old [47].
Table 5.
Comparison of pure RNA yield and purity, obtained from four strains of E. siliculosus by two different methods: the new and old [47].
Figure 2.
Analysis of quality and integrity of extracted nucleic acids.
(A) Genomic DNA and total RNA (∼0.5 µg) isolated simultaneously from four strains of E. siliculosus (RHO12; LIA4A; REP10–11; EC524), using initial biomass of 25, 50 and 100 mg (gel stained with ethidium bromide). DNA shows an intact single band whilst RNA shows the clear cytosolic and plastid (Cp) ribosomal bands. (B) Genomic DNA contamination is effectively removed by DNase treatment, whilst the pure RNA retains intactness and quality. RNA species of low molecular weight are also apparent. M: RNA Ladder, High Range (Fermentas, Italy).
Figure 3.
Gel electrophoresis analysis of pure DNA and its downstream application.
(A) Genomic DNA (∼0.5 µg), after RNase treatment, isolated from strains of E. siliculosus (RHO12, LIA4A; REP10–11; EC524) using initial biomass of 25, 50 and 100 mg. (B) The quality of isolated DNA was confirmed by electrophoresis analysis of a DNA PCR product using an alpha tubuline (TUA) housekeeping gene. (C) Electrophoretic analysis of EcoRV enzyme digestion product of genomic DNA confirms that the extracted DNA is suitable for downstream application (gels stained with ethidium bromide). M: 100-bp, 1-Kb DNA and High Range RNA Ladder (Fermentas, Italy).
Figure 4.
RT-PCR analysis of TUA expression of four strains of E. siliculosus.
RNA samples extracted from four strains of E. siliculosus (RHO12, LIA4A; REP10–11; EC524) using initial biomass of 25, 50 and 100 mg were analyzed by RT-PCR for the alpha tubuline (TUA) housekeeping gene. No amplification was observed when RNA was directly used for PCR (No-RT control panel), indicating that no DNA contamination is present in the RNA starting material. M: 100-bp DNA ladder (Fermentas, Italy); −: PCR negative control (no DNA, but water was added); +: PCR positive control (no RNA but DNA was added).