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Nucleic Acid Purification


Products >  Nucleic_Acid_Purification >  RNA >  MicroRNA >  Tissue

miRNA Purification & Isolation Kit—NucleoSpin miRNA

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

  1. Mechanically disrupt sample material in Lysis Buffer ML.
    Alternatively, perform a phenol/chloroform extraction of RNA from cells/tissues.
  2. Homogenize and clear crude lysates with NucleoSpin Filters (violet rings).
  3. Add ethanol to adjust binding conditions for DNA and large RNA.
  4. Bind DNA and large RNA to the NucleoSpin RNA Column (blue ring).
    The flow-through of the NucleoSpin RNA Column contains miRNA and protein.
  5. Remove residual genomic DNA by on-column digestion with RNase-free recombinant DNase (included in the kit).
  6. Wash large RNA and elute with RNase-free water—large RNA fraction.
  7. Add Protein Precipitation Buffer MP to the flowthrough from the NucleoSpin RNA Column (Step 4) to precipitate the protein.
  8. 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.
  9. Add Binding Buffer MX to adjust the binding conditions for miRNA.
  10. Bind miRNA to the NucleoSpin miRNA Column (green ring).
  11. 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.

At-A-Glance Documents Images & Data Resources

Features

  • Effective isolation of miRNA, other RNA, and total RNA from a variety of samples
  • Effective purification of miRNA and total RNA in two fractions
  • Effective purification of miRNA and total RNA in one fraction
  • Ideal for isolation and purification of miRNA, siRNA, shRNA, snRNA, and other small RNAs
  • Isolate total protein fraction
  • Choice of lysis conditions, with or without phenol/chloroform
  • Complete kit includes all materials

Applications

  • Parallel isolation and purification of miRNA and large RNA from human/animal tissue and cultured cells
  • Parallel isolation and purification of miRNA and large RNA from plant tissue
  • Parallel isolation and purification of miRNA and large RNA in combination with phenol/chloroform (e.g., Trizol) lysis
  • Isolation and purification of miRNA, large RNA, and protein from the same sample
  • Purification of siRNA and large dsRNA from DICER reactions

Downstream Applications

RNA is suitable for:

  • Northern blot analysis
  • Real-time RT-PCR
  • Microarray analysis
  • And more

Product Overview

Technology Silica membrane
Format Mini spin columns
Starting Material <107 cultured cells
<30 mg human/animal tissue
<50 mg plant tissue
<150 µl reaction mixture
Fragment Size – miRNA 18–200 bases
Fragment Size – Large RNA >200 bases
Typical Yield
(from 107 HeLa Cells)
10 µg small RNA
95 µg large RNA
Binding Capacity 200 µg
Elution Volume 30–100 µl
Preparation Time <45 min (6 preps human/animal tissue, miRNA and large RNA)
<35 min (6 preps human/animal tissue, miRNA only)

 
 
Products
Cat. # Product Package Size Price # of Units Select
740971.10 NucleoSpin® miRNA 10 Preps $73.00
740971.50 NucleoSpin® miRNA 50 Preps $312.00
740971.250 NucleoSpin® miRNA 250 Preps $1,367.00
 

 

 

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