| NucleoBond RNA Soil | NucleoBond RNA Soil Mini | |
| Technology | Anion exchange chromatography | |
| Format | Gravity flow columns combined with MN Bead Tubes Type A | |
| Starting material | <2 g of soil | 0.25–0.5 g soil |
| Fragment size | >100 nt | |
| Typical yield | 1–10 µg | 0.25–2.5 µg |
| A260/A280 | 1.7–2.1 | 1.5–2.0 |
| A260/A230 | 1.8–2.5 | — |
| Typical RIN (RNA integrity number) | >8.5 | >7.0 |
| Elution volume | 100 µl | 50–100 µl |
| Preparation time | 60 min/6 preps | 60 min/12 preps |
| Binding capacity | 600 µg | 30 µg |
Product overview
NucleoBond RNA Soil and NucleoBond RNA Soil Mini
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 (MN 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
- Parallel preparation of RNA and DNA in one hour when combined with the DNA Set for NucleoBond RNA Soil or DNA Set for NucleoBond RNA Soil Mini
At-a-glance
High yield and quality
High RNA yields with NucleoBond RNA Soil. Various soil samples (clay, shore soil, forest soil) were purified in duplicate with NucleoBond RNA Soil (MN) using the standard kit protocol. For comparison, the samples were processed in parallel using competitor kit "Q" and the corresponding kit protocol. Each RNA sample was eluted in a volume of 100 μl and yields were determined photometrically (left panel). In addition, 15 µl of each eluate was analyzed by gel electrophoresis (1% TAE; right panel). For each sample type, NucleoBond RNA Soil provided a higher yield compared to the competitor kit.
Superior RT-PCR performance with NucleoBond RNA Soil. Various soil samples (clay, shore soil, and forest soil) were purified in duplicate using NucleoBond RNA Soil (MN) and competitor kit "Q" by following the standard protocol for each kit. RT-PCR was then performed using 40 µl of eluate from each sample (amplicon size = 466 bp). Samples prepared using NucleoBond RNA Soil demonstrated lower CP values relative to corresponding samples prepared using kit "Q".
Parallel DNA isolation
NucleoBond RNA Soil enables efficient parallel DNA isolation. Various soil samples (clay, shore soil, and forest soil) were purified in duplicate using NucleoBond RNA Soil in combination with the DNA Set for NucleoBond RNA Soil (MN) following the standard kit protocol. For comparison, the samples were processed in parallel using competitor kit "Q" and the corresponding protocol. DNA samples were eluted in a volume of 100 µl and yields were determined photometrically (left panel). In addition, 15 µl of each eluate was analyzed by gel electrophoresis (1% TAE; right panel). For each sample type, NucleoBond RNA Soil combined with the DNA Set for NucleoBond RNA Soil provided a higher yield of DNA compared to the competitor kit.
Mini format available
NucleoBond RNA Soil Mini yields sufficient RNA for qPCR analysis. Two different soil samples were purified in duplicate using NucleoBond RNA Soil Mini in comparison with NucleoBond RNA Soil. RNA was eluted in 100 µl and analyzed photometrically, and 15 µl of each eluate was used for gel electrophoresis (1% TAE). RNA yields obtained with NucleoBond RNA Soil Mini were proportional to the reduced input amount for this format relative to yields obtained with NucleoBond RNA Soil, and sufficient for downstream applications such as qRT-PCR.
NucleoBond RNA Soil Mini provides efficient parallel DNA isolation. Two different soil samples were purified in duplicate using NucleoBond RNA Soil Mini in comparison with NucleoBond RNA Soil. DNA was eluted in 100 µl and measured photometrically, and 15 µl of each eluate was applied to a 1% TAE gel for electrophoresis analysis. DNA yields obtained using NucleoBond RNA Soil Mini with the DNA Set for NucleoBond RNA Soil Mini were proportional to the reduced input amount for this format relative to yields obtained with NucleoBond RNA Soil and the corresponding DNA Set for NucleoBond RNA Soil.
Protocol
Protocol overview for NucleoBond RNA Soil*.
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.
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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.
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