Small and large RNA isolation
No toxic phenol / chloroform convenient extraction
Parallel isolation of small and large RNA
• 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 (patent pending)
• NucleoSpin® Filters for efficient sample homogenization
• rDNase for efficient on-column removal of genomic DNA
• Silica-membrane technology
• Sample material: < 107 cultured cells, < 30 mg human / animal tissue, < 50 mg plant tissue, < 150 µL reaction mixture
• Typical yield: 10 µg small RNA, 95 µg large RNA (107 HeLa cells)
- Kit contents :
- NucleoSpin® RNA Columns, NucleoSpin® miRNA Columns, NucleoSpin® Protein Removal Columns, Collection Tubes (2 mL, 2 mL lid, 1.5 mL), NucleoSpin® Filters, buffers, RNase-free rDNase
- Reference numbers :
- 10 preps: 740971.10
- 50 preps: 740971.50
- 250 preps: 740971.250
- 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
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.
- Eur J Hum Genet. 2015 Aug;23(8):1100-5. doi: 10.1038/ejhg.2014.244. Epub 2014 Nov 12.
Expression analysis in intestinal mucosa reveals complex relations among genes under the association peaks in celiac disease.
Plaza-Izurieta L, Fernandez-Jimenez N, Irastorza I