Cytoplasmic and Nuclear RNA Purification Kit
For the convenient purification of cytoplasmic and nuclear RNA from cultured cells and tissues
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For research use only and NOT intended for in vitro diagnostics.
Cytoplasmic and Nuclear RNA Purification Kit
For the convenient purification of cytoplasmic and nuclear RNA from cultured cells and tissues
Overview
- Excellent separation and purification of cytoplasmic and nuclear RNA
- Convenient and fast spin column format
- High quality and yield of RNA
- Isolate full diversity of RNA (including microRNA) without phenol
- Purified RNA is ready for any application including RT-PCR, qRT-PCR, RNA-Seq, arrays and more
- Cytoplasmic RNA is free of DNA and ready for direct use in RT-PCR/qRT-PCR
- Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix
This kit provides a rapid method for the isolation and purification of both cytoplasmic and nuclear RNA from cultured animal cells and small tissue samples. The kit can be used to isolate all sizes of RNA from the cytoplasmic and nuclear RNA fractions, including all small RNA species without any requirement for phenol. Included in the kit are sufficient reagents to perform either 50 cytoplasmic RNA preparations or 25 cytoplasmic and 25 nuclear RNA preparations. Ten samples can be processed in approximately 45 minutes. This kit is also available in a 100 prep size.
Background
In certain circumstances it is desirable to be able to isolate fractionated RNA as opposed to total RNA. For example, it may be preferable to isolate only mature, cytoplasmic RNA for some studies on expression profiling. Alternatively it may be desirable to isolate nuclear RNA in order to investigate and study pre-processed (non-spliced) RNA. Furthermore, this kit can be used to isolate RNA for downstream applications where it is necessary to avoid DNA contamination, since the cytoplasmic fraction has been shown to be free of all traces of genomic DNA.
Details
Supporting Data
Figure 1. Effective Separation of HeLa Cell Cytoplasmic & Nuclear RNA. Norgen's Cytoplasmic & Nuclear RNA Purification Kit was used to effectively separate cytoplasmic and nuclear RNA from 0.75 million HeLa cells in triplicate. Panel A: High quality cytoplasmic and nuclear RNA was purified using Norgen's kit. Note the integrity and quality of both cytoplasmic and nuclear RNA. Ten microliters of each 50 µL elution were run on a 1.5% formaldehyde-agarose gel. Lane M is Norgen's 1 kb RNA Ladder, lanes 1-3 contain nuclear RNA and lanes 4-6 contain cytoplasmic RNA. Panel B: Ten microliters of the above cytoplasmic and nuclear RNA isolated from HeLa cells using Norgen's kit was run on a 0.9% agarose DNA gel. Genomic DNA is clearly visible in the nuclear RNA fractions (lanes 1-3), however, no genomic DNA can be detected in the cytoplasmic RNA fractions (lanes 4-6). Note that an optional on-column DNase treatment protocol is provided to remove the genomic DNA in the nuclear fraction.
Figure 2. Superior Separation of HeLa Cell Cytoplasmic & Nuclear RNA. Norgen's Cytoplasmic & Nuclear RNA Purification Kit provides better separation of cytoplasmic and nuclear RNA from 0.8 million HeLa cells when compared to a leading competitor’s product. Panel A: Cytoplasmic and nuclear RNA purified from HeLa cells using Norgen's kit and a competitor's kit. Ten microliters of each 50 µL elution (Norgen or competitor's kit) of the cytoplasmic (C) or nuclear (N) RNA were run on a 1.5% formaldehyde-agarose gel. Higher yields of RNA with good integrity were isolated using Norgen's kit. Panel B: Ten microliters of the cytoplasmic and nuclear RNA isolated from HeLa cells using Norgen's kit and the competitor's kit was run on a 0.9% agarose gel. Genomic DNA only co-migrates with the nuclear fraction in RNA isolated using Norgen’s Cytoplasmic & Nuclear RNA Purification Kit, not the cytoplasmic fraction. Note that an optional on-column DNase treatment protocol is provided to remove the genomic DNA in the nuclear fraction. In contrast, significant genomic DNA contamination was observed in the cytoplasmic fraction of the RNA isolated using the competitor's kit.
Figure 3. Genomic DNA-free Cytoplasmic RNA. RT-PCR was performed using human beta actin primers on 2 µL of the 50 µL of cytoplasmic RNA isolated from 1 million HeLa cells using Norgen's Cytoplasmic & Nuclear RNA Purification Kit. Lane M is Norgen's PCR Sizer DNA ladder, Lanes 1 and 3 are the negative control (PCR only, without reverse transcript), and Lanes 2 and 4 are the actual RT-PCR that show the expected 166 bp RT-PCR product. The expected amplicon size from the gene copy is the same as that from the RNA transcript. The lack of product in lanes 1 and 3 indicates that no genomic DNA contamination is present in the isolated cytoplasmic RNA. All PCR products were resolved on a 1X TAE, 2% agarose gel.
Figure 4. Specific Separation and Subsequent Amplification of Cytoplasmic and Nuclear Transcripts. Norgen's Cytoplasmic & Nuclear RNA Purification Kit effectively separates cytoplasmic and nuclear RNA. RT-PCR was performed using human U2 snRNA (Panel A) or S14 (Panel B) primers on 0.5 µg of either cytoplasmic or nuclear RNA fractions isolated from 1 million HeLa cells using Norgen's Cytoplasmic & Nuclear RNA Purification Kit. In Panel A, an intense PCR product was observed only in the nuclear fraction when the nuclear-specific U2 snRNA primers were used. In Panel B, the majority of the PCR product for the house-keeping transcript of S14 was observed in the cytoplasmic fraction. The two results combined showed effective separation of the cytoplasmic and nuclear RNA using Norgen's Cytoplasmic & Nuclear RNA Purification Kit. All PCR products were resolved on a 1X TAE, 1.5% agarose DNA gel.
Kit Specifications
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Maximum Column Binding Capacity
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Up to 50 µg RNA
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Maximum Column Loading Volume
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650 µL
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Size of RNA Purified
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All sizes, including small RNA (< 200 nt)
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Time to Complete 10 Purifications |
45 minutes
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RNA Yield
HeLa (1 x 106) - Cytoplasmic RNA HeLa (1 x 106) - Nuclear RNA |
15 µg ≤ 3.5 µg |
Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment.
Component | Cat. 21000 (50 preps) | Cat. 37400 (100 preps) |
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Lysis Buffer J | 20 mL | 2 x 20 mL |
Buffer SK | 40 mL | 2 x 40 mL |
Wash Solution A | 38 mL | 2 x 38 mL |
Elution Buffer E | 6 mL | 2 x 6 mL |
Mini Spin Columns | 50 | 100 |
Collection Tubes | 50 | 100 |
Elution Tubes (1.7 mL) | 50 | 100 |
Product Insert | 1 | 1 |