2Dr. Lerche KG, Berlin, Germany
Materials and methods
Here we report that dehydrated ethanol is an excellent medium for both in situ preservation of nucleic acids and cell disruption of plant and yeast cells. Cell disruption was strongly facilitated by prior dehydration of the ethanol using dehydrated zeolite. Following removal of ethanol, nucleic acids were extracted from the homogenate pellet using denaturing buffers. The method provided DNA and RNA of high yield and integrity. Whereas cell wall disruption was essential for extraction of DNA and large RNA molecules, smaller molecules such as tRNAs could be selectively extracted from undisrupted, ethanol-treated yeast cells. Our results demonstrate the utility of absolute ethanol for sample fixation, cell membrane and cell wall disruption, as well as preservation of nucleic acids during sample storage.
Cell disruption in ethanol has long been used for the isolation of cell wall material from plants (1). Although not applicable to the isolation of native proteins or organelles, cell disruption in ethanol might be suitable for isolation of nucleic acids. Reports on long-term in situ preservation of DNA in ethanol are controversial. Whereas initial attempts to fix plant tissues in ethanol or other organic solvents showed rapid decay of DNA (2,3), 95% or 100% ethanol has more recently been successfully used for the in situ preservation of DNA in plant tissues (4,5). The aim of the current study was to reevaluate the suitability of ethanol as a preserving medium for RNA and DNA, especially during the mechanical disruption of biological samples with rigid cell walls, such as yeast and plant cells.
When yeast cells (complete materials and methods can be found in the Supplementary Materials) were ruptured in an aqueous buffer with zirconium oxide beads as previously described (6), complete cell disruption was not reached within 1 h. The mechanical force generated by vortex mixing with glass beads was insufficient to disrupt yeast cells in the solubilizing buffer or in 90% ethanol. However, in 96% ethanol, the majority of the cells were broken in ≤5 min when using the same homogenization procedure. Reduction of the water activity to near zero by means of zeolite beads markedly increased the homogenization efficiency (Figure 1, A–C).
When immersed overnight in absolute ethanol together with the zeolite beads, plant materials became brittle and could be completely disrupted with the zirconium oxide beads on the vortex shaker (Figure 1D). Comparable disruption required at least a 2-fold longer amount of time when performed without zeolite pretreatment (data not shown). The negative effect of a low water concentration is most likely the result of the cell wall polymers’ high affinity for water. When water in the cell wall is completely removed, the scaffolds of fungal and plant cells walls become stiffer, and mechanical impulses are no longer efficiently damped by elastic deformation. While disruption with beads in ethanol is well-suited to microbial cells and thin plant structures, other techniques for fixation and disruption with ethanol might be necessary when working with bulkier plant tissues. Fixation and preservation in ethanol of thin leaves or small flowers is easy and safe. This is an important advantage relative to RNAlater (U.S. patent no. 6204375), since these plant materials tend to float upon that solution and be slowly immersed in it.
When yeast cells were disrupted in ethanol and extracted using the protocol described in the Supplementary Materials, the extracts contained high molecular weight DNA and high amounts of RNA (Figure 2A). In contrast, destruction of the lipid membranes by ethanol without cell disruption allowed for the subsequent extraction of small RNAs (Figure 2, B–D). Therefore, the sharp size permeation limit of the yeast cell wall (7-10) may be useful for selective extraction of small RNA molecules. It is noteworthy that yeast cell walls were partly disrupted when complete dehydration was carried out by slow shaking of the cells with the zeolite beads. In this case, rRNA and DNA appeared in the extracts (Figure 2E). Complete dehydration alone did not give this result (Figure 2F). For plant materials completely disrupted in ethanol, the extracts contained well-preserved RNA and DNA (Figure 2, G–M). After storage in ethanol for 1–2 months, high molecular weight nucleic acids remained extractable from mechanically disrupted yeast cells (data not shown) and plant tissues (Figure 2, I–L).
The yield of DNA extracted from yeast reached ~1%, and the yield of total nucleic acids was ~10% of dry weight (Supplementary Table S1). These yields are comparable to those of previous reports using dormant baker's yeast (11). An extraction time of 15 min was sufficient (Supplementary Table S2). Lower yields were obtained at an SDS concentration of 0.7%, with a 1.4% SDS concentration sufficient to obtain maximum yields if the ethanol was completely evaporated. Complete drying of the homogenate pellet from anhydrous ethanol was possible without loss of extractability. When homogenate pellets were dried completely from ethanol containing water, they were not easily dissolvable in the extraction buffers. From Supplementary Tables S1 and S2, it may be derived that storage of yeast cell homogenates for 1 month in absolute ethanol at room temperature does not affect yield.
RNA and DNA could be extracted and purified from the dried cell homogenate by standard protocols (see the Supplementary Methods). After removal or dilution of SDS, the DNA could be used for PCR (Figure 3A). Digestion patterns obtained using EcoRI, EcoRV, or PvuII (Figure 3B) demonstrate the suitability of column-purified DNA for applications requiring digested DNA samples (e.g., RFLP, AFLP analysis). Southern blot analysis of extracted yeast DNA using a 320-bp probe from the first exon of the mitochondrial gene cytochromec oxidase II (COXII; V00685) is shown. Two distinct bands of size 1.97 and 0.27 kb can be seen, corresponding to the restriction fragment lengths deduced from the sequence of the mitochondrial genome. This provides further evidence of the DNA quality of the extracted samples.
RNA quality was visualized by agarose gel electrophoresis under denaturing conditions, showing a ratio of the intensities of 28S to 18S rRNA bands to be ~2 (Figure 3C, lanes 1 and 2). The quality of RNA in column-purified SDS extracts from Saccharomyces cerevisiae was high, as demonstrated by RNA integrity number (RIN) values of 9.0–9.2. Purification using acidguanidinium thiocyanate-phenol-chloroform treatment typically resulted in RIN values of 8.0–8.8. Generally, RIN values of 8.0–9.2 reflect an RNA quality sufficient for downstream molecular applications. As shown for Arabidopsis, Daucus, and Saccharomyces, the purified RNA could be used for RT-PCR (Figure 3C, lanes 3–10; see the Supplementary Methods).
We have demonstrated that completely dehydrated cells and tissues are more easily disrupted in ethanol than hydrated ones, and the homogenates provided high yields of DNA and RNA with good structural integrity suitable for analyses using standard molecular biological methods. Furthermore, we showed the described method of cell disruption is compatible with established methods of RNA and DNA extraction and purification. The combination of efficient cell disruption with in situ preservation of RNA and DNA in ethanol offers an attractive alternative to both sample storage in the RNAlater solution and grinding with liquid nitrogen.
We thank Thomas John Buckhout for discussions and critical reading of the manuscript and Ekkehard Richter for support in microphotography. Financial support of this study by the program “Wirtschaft trifft Wissenschaft” of the Bundesministerium fur Verkehr, Bau, und Stadtentwicklung (project no. 03WWBE061) is gratefully acknowledged.
The authors declare no competing interests.
Address correspondence to Rudolf Ehwald, Department of Biology, Cell Biology, Humboldt University, Invalidenstr. 42, D-10115 Berlin, Germany. e-mail: [email protected]