microRNA extraction without phenol/chloroform
Principles
NucleoSpin miRNA is suitable for simultaneous isolation of small RNA (< 200 nt, e.g., miRNA, pre-miRNA, tRNA, 5S RNA), large RNA (> 200 nt, e.g., mRNA, 18S rRNA, 28S rRNA, pri-miRNA), and protein in three separate fractions from a large variety of sample materials:
- 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 flow-through 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 of large RNA and elution with RNase-free water > large RNA fraction.
- Addition of Protein Precipitation Buffer MP to the flow-through of the NucleoSpin RNA Column (step 4) to precipitate the protein.
- Collection of protein precipitate by centrifugation > protein fraction.
Filtration of the supernatant through a NucleoSpin Protein Removal Column (white ring) to completely remove the residual protein precipitate to achieve best possible purity of the small RNA fraction.
The flow-through of the NucleoSpin Protein Removal Column only contains 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 with RNase-free water > small RNA fraction.
The precipitated protein can easily be dissolved in Laemmli buffer and used for SDS-PAGE, Western Blot analysis, and protein quantification, for example with the MACHEREY-NAGEL Protein Quantification Assay. The eluted RNA and miRNA are ready-to-use for all standard downstream applications, for example RT-PCR, Northern Blot, or chip hybridization.
NucleoSpin miRNA procedure
