Cytoplasmic & Nuclear RNA Purification Kit

Cytoplasmic & Nuclear RNA Purification Kit

Catalog# : NB-21000
Price/Unit : $295.00
Unit : 50 preps
For the convenient purification of cytoplasmic and nuclear RNA from cultured cells and tissues

* High quality RNA
* No need for phenol
* Genomic DNA-free cytoplasmic RNA

Bio-Synthesis's Cytoplasmic & Nuclear RNA Purification Kit provides a rapid method for the isolation and purification of both cytoplasmic and nuclear RNA from cultured animal cells and small tissue samples in as little as 45 minutes. In certain circumstances it is desirable 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, or nuclear RNA in order to study pre-processed 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.

Purification is based on spin column chromatography using Bio-Synthesis's proprietary resin as the separation matrix. Briefly, the process involves first lysing the cells or tissue of interest with the provided Lysis Solution. The lysate is then separated through centrifugation, with the supernatant containing the cytoplasmic RNA and the pellet containing the nuclear RNA. Binding solution and ethanol are then added to the desired fraction, and the solution is loaded onto a spin-column (BIND 1 and BIND 2). Under these conditions only the RNA will bind to the column, while the contaminating proteins will be removed in the flowthrough or retained on the top of the resin. The bound RNA is then washed with the provided Wash Solution in order to remove any remaining impurities (WASH 1 and WASH 2). Lastly, the desired purified RNA is eluted into 50 µL of the provided Elution Buffer or water (ELUTE 1 and ELUTE 2). Please see the procedure flowchart to the right.

The purified RNA is of the highest integrity, and can be used in a number of downstream applications including real time PCR, reverse transcription PCR, Northern blotting, RNase protection and primer extension, expression array assays, miRNA cloning and Next Generation Sequencing.



FEATURES AND BENEFITS

* Fast and easy processing - Rapid spin-column format allows for the processing of 10 samples in 45 minutes.
* No genomic DNA contamination in cytoplasmic fraction - Purified cytoplasmic RNA can be used directly in RT-PCR reactions with no DNAse treatment required.
* No phenol:chloroform extractions- Cytoplasmic and nuclear RNA is isolated without the use of harmful chemicals such as phenol or chloroform.
* Isolate a diversity of RNA species - All nuclear and cytoplasmic RNA species can be isolated, from large mRNA species down to microRNA (miRNA) and small interfering RNA (siRNA).
* High quality RNA suitable for use in downstream applications - Purified RNA can be used in a number of downstream applications including real-time PCR, reverse transcription PCR, Northern blotting, RNAse protection and primer extension, and expression array analysis requiring the use of intact RNA.

APPLICATIONS

* Quantitative, real-time RT-PCR for both large mRNA and small RNA including miRNA
* RT-PCR for both large mRNA and small RNA including miRNA
* Northern blotting
* RNase protection
* Primer extension
* Expression array assays
* Next Generation Sequencing for RNA and miRNA
* microRNA Cloning


Figure 1. Effective Separation of HeLa Cell Cytoplasmic & Nuclear RNA. Bio-Synthesis'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 Bio-Synthesis'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 Bio-Synthesis'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 Bio-Synthesis'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. Bio-Synthesis'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: High quality cytoplasmic and nuclear RNA from HeLa cells purified using Bio-Synthesis's kit. Note the integrity and quality of both cytoplasmic and nuclear RNA. Ten microliters of each 50 µL elution (Bio-Synthesis 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 Bio-Synthesis's kit . Panel B: Ten microliters of the cytoplasmic and nuclear RNA isolated from HeLa cells using Bio-Synthesis'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 Bio-Synthesis'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 Bio-Synthesis's Cytoplasmic & Nuclear RNA Purification Kit. Lane M is Bio-Synthesis's PCRSizer 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 the that from the RNA transcript. The lack of a product in lanes 1 and 3 indicate that no genomic DNA contamination is present in the isolated cytoplasmic RNA. All PCR products were resolved on a 1X TAE, 2% agarose gel.


Cytoplasmic and Nuclear RNA Purification Kit Components
1. Lysis Solution
2. Binding Solution
3. Wash Solution
4. Elution Buffer
5. Mini Spin Columns
6. Collection Tubes
7. Elution Tubes
8. Product Insert
Long-Term Storage : All solutions should be kept tightly sealed and stored at room temperature. These reagents should remain stable for at least 1 year in their unopened containers.
Product Usage
For laboratory research use only, not for diagnostic use! Not for use in humans.