| Technology | Silica-membrane technology |
| Format | Mini spin columns |
| Sample material | ≤200 µl phenol/chloroform extract, reaction mixture, or <105 cells; <300 ul RNA sample containing <90 ug RNA (XS) |
| Fragment size | >200 nt |
| Typical recovery | 85–95% |
| A260/A280 | 1.9–2.1 |
| Typical RIN (RNA integrity number) | Depends on sample quality (no significant loss of RIN detected) |
| Elution volume | 40–120 µl; 5–30 µl (XS) |
| Preparation time | 20 min/6 preps |
| Binding capacity | 200 µg; 110 µg (XS) |
PRODUCT OVERVIEW
NucleoSpin RNA Clean-up
- Complete removal of RT-PCR inhibitors
- Time-saving procedure based on NucleoSpin RNA, without DNase digestions and homogenization steps
- RNA cleanup from pre-purified RNA (phenol/chloroform), enzymatic reactions (e.g., amplification reactions, labeling reactions)
- For isolation of RNA from cells and tissue with minimal DNA contamination, we recommend rDNase-containing NucleoSpin RNA kits
- XS format available for extra-small sample sizes (see specifications in the At-a-glance table)
At-a-glance
Applications
RNA clean-up for:
- Pre-purified RNA (e.g., Trizol)
- Reaction mixtures
- Amino-allyl-mRNA
- Biotinylated RNA
- RNA isolation from up to 105 cultured cells (whenever co-purification of some genomic DNA is acceptable, kit does not contain rDNase)
- Typical downstream applications: enzymatic-labeling reactions, RT-PCR, DNA/RNA-based chip hybridizations
NucleoSpin RNA Clean-Up kits are recommended for the cleanup of total RNA from RNA preparations which contain unacceptable amounts of RT-PCR inhibitors, such as RNA prepared with phenol-chloroform based methods. These kits are also suitable for the isolation of RNA from small amounts of cultured cells whenever copurification of some genomic DNA is acceptable. The kits allow purification of pure RNA with an A260/A280 ratio generally exceeding 1.9 (measured in TE buffer, pH 7.5).
NucleoSpin RNA Clean-Up kits are also recommended for cleanup of in vitro-transcribed RNA and aminoallyl mRNA, as well as biotinylated and fluorescently-labeled RNA. Purified RNA is ready for use in applications such as enzymatic labeling reactions (including dye incorporation), RT-PCR, and DNA/RNA-based chip hybridizations.
The integrity of purified RNA can be examined by denaturing agarose gel electrophoresis.
Protocol overview
RNA-containing samples are mixed with a solution containing large amounts of chaotropic ions. This solution immediately inactivates RNases—which are present in virtually all biological materials—and creates appropriate binding conditions which favor adsorption of RNA to the silica membrane. Simple washing steps remove salts, metabolites, organics like phenol, and cellular debris. Pure RNA can then be eluted with RNase-free water.
NucleoSpin RNA Clean-Up kit procedure.
Application data
The cRNA purified with NucleoSpin RNA Clean-up Kit from Macherey-Nagel is very suitable for downstream biochip technology and detection using the products from Affymetrix, and gives very excellent results.
—Guido Krupp, Ph.D., Director and Founder, artus GmbH, Hamburg, Germany
Amino-allyl-modified cRNA (total RNA from rat kidney) was purified using either NucleoSpin RNA Clean-Up (blue) or using a kit from competitor Q (red). 1/20 μl was analyzed by Agilent Bioanalyzer 2100 (green: RNA 6000 ladder, Thermo Fisher Scientific).
XS format
Purification of high-quality RNA from reaction mixtures
RNA quality (RIN) and quantity were analyzed with an Agilent® Bioanalyzer, Agilent RNA 6000 Nano chip, and Agilent RNA 6000 Nano reagent before and after clean-up. The crude RNA solution (lanes 1 and 2) show low RNA concentrations around 85 ng/μL. Nevertheless, the RNA is not degraded and has a high RIN of about 9.4. Lanes 3 and 4 show the corresponding RNA after DNase digestion and purification with NucleoSpin RNA Clean-up XS. RNA quality is not affected by the whole procedure, showing similar RIN and high recovery of about 95% in a total volume of 20 μL.
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