NucleoSpin miRNA is suitable for the simultaneous isolation and purification of small RNA (<200 nt, e.g., miRNA, pre-miRNA, tRNA, 5S RNA), large RNA (>200 nt, e.g., mRNA, 18S rRNA, 28S rRNA, pre-miRNA), and protein in three separate fractions from a large variety of sample materials.
Protocol Overview
- Mechanically disrupt sample material in Lysis Buffer ML.
Alternatively, perform a phenol/chloroform extraction of RNA from cells/tissues. - Homogenize and clear crude lysates with NucleoSpin Filters (violet rings).
- Add ethanol to adjust binding conditions for DNA and large RNA.
- Bind DNA and large RNA to the NucleoSpin RNA Column (blue ring).
The flow-through of the NucleoSpin RNA Column contains miRNA and protein. - Remove residual genomic DNA by on-column digestion with RNase-free recombinant DNase (included in the kit).
- Wash large RNA and elute with RNase-free water—large RNA fraction.
- Add Protein Precipitation Buffer MP to the flowthrough from the NucleoSpin RNA Column (Step 4) to precipitate the protein.
- Collect protein precipitate by centrifugation—protein fraction.
Filter the supernatant through a NucleoSpin Protein Removal Column (white ring) to completely remove the residual protein precipitate and achieve the best possible purity of the miRNA fraction.
The flowthrough of the NucleoSpin Protein Removal Column only contains miRNA. - Add Binding Buffer MX to adjust the binding conditions for miRNA.
- Bind miRNA to the NucleoSpin miRNA Column (green ring).
- Wash miRNA and elute with RNase-free water—miRNA fraction.
The precipitated protein can easily be dissolved in Laemmli buffer and used for SDS-PAGE, Western Blot analysis, and protein quantification with the MACHEREY-NAGEL Protein Quantification Assay. The eluted RNA and miRNA are ready-to-use for all standard downstream applications, such as RT-PCR, Northern blotting, and chip hybridization.
