Metagenomic analyses of soil and sediment samples are often hampered by insufficient lysis of microbial cells and the relatively low amounts of RNA present in these sample types. Developed with these challenges in mind, the NucleoBond RNA Soil kit accommodates sample inputs as large as 2 g and employs ceramic beads (NucleoSpin Bead Tubes Type A) to aid in processing of hard-to-lyse microorganisms, typically yielding 1–10 µg of high-quality RNA per sample. For greater experimental flexibility, NucleoBond RNA Soil Mini provides the same purification technology in a smaller format.
Anion exchange technology to optimize RNA yield and purity—suitable for metagenomic studies
Combination of mechanical homogenization and chemical lysis allows processing of large sample amounts
Gravity flow columns combined with NucleoSpin Bead Tubes Type A
<2 g of soil
0.25–0.5 g soil
Typical RIN (RNA integrity number)
60 min/6 preps
60 min/12 preps
High yield and quality
Parallel DNA isolation
Mini format available
Organisms in each soil sample are lysed by bead beating in the presence of Lysis Buffer E1 and phenol:chloroform:isoamyl alcohol. Buffer OPT can be included in the lysis step to reduce the adsorption of nucleic acids to clay and mineral soil components but will also increase contamination with humic acids (if present). Performance cannot be predicted and depends on the soil composition, but as a rule of thumb a sample with a high content of organic and humic components (e.g., forest soil) should be lysed without Buffer OPT while predominantly mineral soils (e.g., river sediments and clay) should be lysed with Buffer OPT. Unlysed components are sedimented by centrifugation.
In the protocol for NucleoBond RNA Soil, the supernatants of the four bead tubes are transferred and combined into one fresh 15-ml tube and mixed with Binding Buffer E2. The incubation and centrifugation steps that follow can be used to prepare the NucleoBond RNA Columns. The columns including filters are fixed with the supplied Plastic Washers on top of a 50-ml tube or laboratory flask or any NucleoBond Rack. Filters and columns are equilibrated by Buffer EQU to pre-wet the filters and to prepare the anion exchange chromatography columns. Once the columns are equilibrated, the clear supernatant of the centrifuged samples is loaded onto the filters in the purification columns. Undissolved particles and some of the soluble macro molecules will be held back by the filters. Wash Buffer E3 is used to flush the filter columns and to wash the nucleic acids from the filters onto the column matrix. Nucleic acids and soluble polyanionic molecules including some fraction of humic substances (if present) will bind to the anion exchange surface. A brown layer might be visible on top of the silica matrix. After the washing step, the filter is removed, and the anion exchange column is washed with Buffer E4 to remove contaminants. RNA is eluted by Buffer ERNA. If isolation of RNA and DNA is desired, DNA can be eluted with Buffer EDNA, included with the DNA Set for NucleoBond RNA Soil (Cat. # 740141.20 available separately; see below) from the same column. Isopropanol is added to the eluates and the mixture is bound to NucleoSpin Finisher Columns by centrifugation. The columns are washed with Buffer E5 and dried. Samples are then eluted in RNase-free H2O.
*NucleoBond RNA Soil Mini uses a similar protocol, but on a smaller scale.
The better way to isolate RNA
Critical factors when purifying RNA are the complete removal of contaminating DNA (gDNA) and the immediate prevention of degradation of RNA. The NucleoSpin RNA Plus kit introduces the NucleoSpin gDNA Removal Column, a spin column which quickly and completely removes genomic DNA contamination without the need for DNase digestion. To maintain RNA integrity, cells and tissues are first lysed by incubation in a chaotropic ion lysis buffer solution, which immediately inactivates RNases.