|Format||Mini spin columns|
<107 cultured cells
|Fragment size, small RNA
|Fragment size, large RNA||>200 bases|
10 µg small RNA, 95 µg large RNA from 107 HeLa cells
|Binding capacity||200 µg|
|Elution volume||30–100 µl|
|Preparation time||<45 min (6 preps human/animal tissue, small and large RNA)
<35 min (6 preps human/animal tissue, small RNA only)
Nucleic acid purification
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Parallel isolation of small RNA, large RNA, and protein
The NucleoSpin miRNA kit is designed for the simultaneous isolation of small RNA, such as miRNA (<200 nt), large RNA (>200 nt), and protein in separate fractions and without the use of phenol/chloroform. The kit accommodates a large variety of sample inputs including cultured cells, human/animal tissue, plant samples, and reaction mixtures. For isolation of miRNA from bodily fluids (blood, plasma, serum, saliva, urine, etc.), we instead recommend the use of the NucleoSpin miRNA plasma kit.
Key characteristics and components of NucleoSpin miRNA kit include:
- Parallel isolation of small RNA, large RNA, and protein
- RNA purification fractionated by size:
- Isolation of small RNA only (<200 nt)
- Isolation of small RNA (<200 nt) and large RNA (>200 nt) in two separate fractions
- Isolation of total RNA (small and large RNA) in a single combined fraction
- Additional isolation of a total protein fraction, ready to use in SDS-PAGE and Western blot analyses
- Excellent RNA yield and purity through lysis with chaotropic salts (no phenol/chloroform)
- NucleoSpin Filters for efficient sample homogenization
- rDNase for efficient on-column removal of genomic DNA
Isolation of small and large RNA
High RNA yield and purity
High yield from small, large, and total RNA fractions
|Sample material||Amount||Protocol used||Yield total
|Mouse liver||30 mg||Tissue||100||105||19|
|Mouse kidney||30 mg||Tissue||35||31||9|
|Mouse spleen||30 mg||Tissue||48||36||22|
|Mouse lung||30 mg||Tissue||27||21||9|
|Mouse heart||30 mg||Trizol||24||19||4|
|Porcine liver||30 mg||Tissue||80||70||13|
|Human brain||30 mg||Tissue||11||10||3|
|Human brain||30 mg||Trizol||17||14||3|
|HeLa cells||107 cells||Cells||100||100||10|
Linear relationship between input cell numbers and RNA yield
High yield of siRNA and dsRNA fractions
Efficient removal of genomic DNA contamination
SDS-PAGE of protein fraction
- 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 flowthrough 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 and elution of large RNA fraction with RNase-free water.
- Addition of Protein Precipitation Buffer MP to the flowthrough of the NucleoSpin RNA Column (step 4) to precipitate the protein.
- Collection of protein precipitate by centrifuging the protein fraction.
- Filtration of the supernatant through a NucleoSpin Protein Removal Column (white ring) to completely remove the residual protein precipitate and improve purity of the small RNA fraction. The flowthrough of the NucleoSpin Protein Removal Column contains only 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 of small RNA fraction with RNase-free water.
The precipitated protein can easily be dissolved in Laemmli buffer and used for SDS-PAGE, Western blot analysis, and protein quantification.
The eluted RNA and miRNA are ready-to-use for all standard downstream applications, for example RT-PCR, Northern blot, or chip hybridization.
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