MACHEREY-NAGEL 740971 NucleoSpin miRNA
Parallel isolation of small and large RNA
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.
||Mini spin columns
||< 107 cultured cells, < 30 mg human / animal tissue
< 50 mg plant tissue, < 150 µl reaction mixture
||size Small RNA: < 200 b, large RNA: > 200 b
||10 µg small RNA, 95 µg large RNA from 107 HeLa cells
||< 45 min (6 preps human / animal tissue, small and large RNA)
< 35 min (6 preps human / animal tissue, small RNA only)
- RNA purification fractionated by size: Isolation of small RNA only (< 200 b),
Isolation of small RNA (< 200 b) and large RNA (> 200 b) in two separate fractions
Isolation of total RNA (small and large RNA in one fraction)
- Additional isolation of total protein fraction ready to use for SDS-PAGE and Western blot analysis
- Excellent RNA recovery and purity by chaotropic salt lysis without phenol/chloroform
- NucleoSpin® Filters for efficient sample homogenization
- rDNase for efficient on-column removal of genomic DNA
- Parallel isolation of small and large RNA from human / animal tissue and cultured cells, from plant tissue, and in combination with phenol / chloroform (e.g., TRIzol®) lysis
- Purification of siRNA and large dsRNA from DICER reactions
- Typical downstream applications: real-time RT-PCR, Northern blotting, chip hybridization
Kits to be used for research purposes only
|Manual||Isolation of small and large RNA||download|
|Brochure / Flyer||RNA Purification Products||download||