Para la purificación de ARN citoplasmático y nuclear de células cultivadas y tejidos
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.
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Especificaciones del kit
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Capacidad máxima de unión de la columna;
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hasta 50 µg de ARN
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Volumen máximo de carga de la columna
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650 µL
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Tamaño del ARN purificado
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Todos los tamaños, incluido el ARN pequeño (< 200 nt)
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| Tiempo para completar 10 purificaciones |
45 minutos
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Rendimiento del ARN
HeLa (1 x 106) - ARN citoplasmático HeLa (1 x 106) - ARN nuclear |
15 µg ≤ 3.5 µg |
Condiciones de almacenamiento y estabilidad del producto
Todas las soluciones deben mantenerse herméticamente cerradas y conservarse a temperatura ambiente. Este kit es estable durante 2 años a partir de la fecha de envío.