| Technology | Silica membrane |
| Format | Mini spin columns |
| Sample size |
<107 cultured cells |
| Fragment size, small RNA |
18–200 bases |
| Fragment size, large RNA | >200 bases |
| Typical yield |
10 µg small RNA, 95 µg large RNA from 107 HeLa cells |
| Binding capacity | 200 µg |
| Elution volume | 30–100 µl |
| Preparation time | <45 min (6 preps human/animal tissue, small and large RNA) <35 min (6 preps human/animal tissue, small RNA only) |
Product overview
Parallel isolation of small RNA, large RNA, and protein
The NucleoSpin miRNA kit is designed for the simultaneous isolation of small RNA, such as miRNA (<200 nt), large RNA (>200 nt), and protein in separate fractions and without the use of phenol/chloroform. The kit accommodates a large variety of sample inputs including cultured cells, human/animal tissue, plant samples, and reaction mixtures. For isolation of miRNA from bodily fluids (blood, plasma, serum, saliva, urine, etc.), we instead recommend the use of the NucleoSpin miRNA plasma kit.
Key characteristics and components of NucleoSpin miRNA kit include:
- Parallel isolation of small RNA, large RNA, and protein
- RNA purification fractionated by size:
- Isolation of small RNA only (<200 nt)
- Isolation of small RNA (<200 nt) and large RNA (>200 nt) in two separate fractions
- Isolation of total RNA (small and large RNA) in a single combined fraction
- Additional isolation of a total protein fraction, ready to use in SDS-PAGE and Western blot analyses
- Excellent RNA yield and purity through lysis with chaotropic salts (no phenol/chloroform)
- NucleoSpin Filters for efficient sample homogenization
- rDNase for efficient on-column removal of genomic DNA
Product summary
Isolation of small and large RNA
Figure 1. Extraction of small and large RNA populations. Panel A. Total RNA was isolated from 107 HeLa cells using NucleoSpin miRNA (orange) and two competitor kits based on phenol/chloroform lysis and extraction (green) or phenol/chloroform extraction (blue). Equal amounts of total RNA fractions were analyzed on an Agilent Bioanalyzer. The NucleoSpin miRNA kit can efficiently isolate both small (Panel B) and large (Panel C) RNA in separate fractions with very high recovery and without phenol/chloroform.
High RNA yield and purity
High yield from small, large, and total RNA fractions
| Sample material | Amount | Protocol used | Yield total RNA (µg) |
Yield large RNA (µg) |
Yield small RNA (µg) |
| Mouse liver | 30 mg | Tissue | 100 | 105 | 19 |
| Mouse kidney | 30 mg | Tissue | 35 | 31 | 9 |
| Mouse spleen | 30 mg | Tissue | 48 | 36 | 22 |
| Mouse lung | 30 mg | Tissue | 27 | 21 | 9 |
| Mouse heart | 30 mg | Trizol | 24 | 19 | 4 |
| Porcine liver | 30 mg | Tissue | 80 | 70 | 13 |
| Human brain | 30 mg | Tissue | 11 | 10 | 3 |
| Human brain | 30 mg | Trizol | 17 | 14 | 3 |
| HeLa cells | 107 cells | Cells | 100 | 100 | 10 |
Table I. Total RNA and fractionated RNA from the indicated maximum recommended amounts of different sample materials were purified with NucleoSpin miRNA according to the individual protocols. Note that RNA yields as well as the ratio of small to large RNA vary due to species, developmental stage, etc. Cultured cells or soft tissue like liver, kidney, lung, etc., can easily be processed with the phenol-free standard procedures. For lipid tissue like brain or very hard-to-lyse, fibrous samples (e.g., heart tissue) it might be advantageous to use the protocol for RNA purification in combination with phenol/chloroform lysis to obtain optimal yields.
Linear relationship between input cell numbers and RNA yield
Figure 2. Direct linear correlation of input cells to RNA yield shown by quantitative RT-PCR. Total RNA was purified from 104, 105, 106, and 107 HeLa cells using NucleoSpin miRNA. MiR-16 was amplified in a qRT-PCR reaction using a Roche LightCycler. The graph shows a perfect linear correlation of input cells and RNA yield. The data table additionally shows corresponding linear decrease of Ct values with increasing RNA input.
High yield of siRNA and dsRNA fractions
Figure 3. Purification of siRNA from a DICER reaction mixture. 2.5, 5, 10, and 15 µg of a double stranded RNA template were digested using DICER (digestion efficiency was 60%). The small siRNA product was purified from the reaction mixture by separating the uncut large dsRNA using the NucleoSpin miRNA clean-up protocol for DICER reactions. The purified siRNA and large dsRNA fractions were analyzed on a 1% TAE agarose gel. The yields of siRNA in lane 9 and large dsRNA in lane 5 correspond to the input amount (lanes 2 and 11) and show the 95% recovery.
Efficient removal of genomic DNA contamination
Figure 4. Highly efficient removal of genomic DNA by on-column DNase digestion. Total RNA from 107 HeLa cells was purified with NucleoSpin miRNA (red) and two competitor kits Q (blue) and A (green). The RNA was assayed for residual traces of DNA by amplifying a 200-bp fragment of the ATPase 6 gene. The ΔCts of 3.5 and 4.3 between NucleoSpin miRNA and competitor A or Q, respectively, indicate a more than tenfold increase in DNA removal by the NucleoSpin miRNA on-column DNase digestion compared to standard phenol/chloroform extractions.
SDS-PAGE of protein fraction
Figure 5. Total protein from various tissues (see Table I) was isolated during the NucleoSpin miRNA procedure. The protein precipitate was dissolved in Laemmli-like protein solubilization buffer PSB and 40 μg was run on a 12% SDS polyacrylamide gel (100 V, 45 min).
Protocol
- Mechanical disruption of the sample material in Lysis Buffer ML.
- Convenient homogenization and clearing of crude lysates with NucleoSpin Filters (violet rings).
- Addition of ethanol to adjust binding conditions for DNA and large RNA.
- Binding of DNA and large RNA to the NucleoSpin RNA Column (blue ring). The flowthrough of the NucleoSpin RNA Column contains small RNA and protein.
- Removal of residual genomic DNA by on-column digestion with the provided RNase-free recombinant DNase.
- Washing and elution of large RNA fraction with RNase-free water.
- Addition of Protein Precipitation Buffer MP to the flowthrough of the NucleoSpin RNA Column (step 4) to precipitate the protein.
- Collection of protein precipitate by centrifuging the protein fraction.
- Filtration of the supernatant through a NucleoSpin Protein Removal Column (white ring) to completely remove the residual protein precipitate and improve purity of the small RNA fraction. The flowthrough of the NucleoSpin Protein Removal Column contains only small RNA.
- Addition of Binding Buffer MX to adjust binding conditions for small RNA.
- Binding of small RNA to the NucleoSpin miRNA Column (green ring).
- Washing of small RNA and elution of small RNA fraction with RNase-free water.
The precipitated protein can easily be dissolved in Laemmli buffer and used for SDS-PAGE, Western blot analysis, and protein quantification.
The eluted RNA and miRNA are ready-to-use for all standard downstream applications, for example RT-PCR, Northern blot, or chip hybridization.
Figure 6. Schematic of workflow for the purification of small (S) and/or large (L) RNA fractions and protein using the NucleoSpin miRNA kit.
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