Cytoplasmic & Nuclear RNA Purification Kit

Supplier
Norgen Biotek
Cat. no.
1021000
Kit size
50
Links
Norgen Biotek Protocol Information sheet

Features

• Rapid spin-column format allows for the processing of 10 samples in 45 minutes.
• Purified cytoplasmic RNA can be used directly in RT-PCR reactions with no DNAse treatment required
• Cytoplasmic and nuclear RNA is isolated without the use of harmful chemicals such as phenol or chloroform.
• All nuclear and cytoplasmic RNA species can be isolated, from large mRNA species down to microRNA (miRNA) and small interfering RNA (siRNA).
• 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.

Norgen'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 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. Norgen’s Cytoplasmic & Nuclear RNA Purification Kit can be used to isolate all sizes of RNA from the cytoplasmic and nuclear RNA fractions, including all small RNA species. The kit is supplied with sufficient reagents to perform either 50 cytoplasmic RNA preparations or 25 cytoplasmic and 25 nuclear RNA preparations.

Purification is based on spin column chromatography using Norgen’s proprietary resin as the separation matrix. The cytoplasmic RNA is preferentially purified from the nuclear RNA and other cellular components such as proteins, without the use of phenol or chloroform. The process involves first lysing the cells or tissue of interest with the provided Lysis Solution (please see the flow chart on page 4). 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. Norgen’s resin binds RNA in a manner that depends on ionic concentrations. Thus 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, and the purified RNA is eluted with the Elution Buffer. 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, and expression array assays.

Column Binding Capacity	50 µg
Maximum Column Loading Volume	600 µl
Size of RNA purified	All sizes, including small RNA (<200 nt)
Maximum Amount of Starting Material:	Animal Cells: 3x10exp6 cells
Time to Complete 10 Purifications	45 minutes
Average Yields*	HeLa Cells – cytoplasmic (1 x 10exp6 cells): 15 µg	HeLa Cells – nuclear (1 x 10exp6 cells): <=3.5 µg	* average yields will vary depending upon a number of factors including species, growth conditions used and developmental stage.

HeLa Cell Cytoplasmic & Nuclear RNA:

High Quality Cytoplasmic & Nuclear RNA Purified Using Norgen's Kit. Note the integrity and quality of both cytoplasmic and nuclear RNA. Ten microliters of each elution were run on a 1.5% formaldehyde-agarose gel. Lane M is Norgen's RNA Ladder, lanes 1-3 contain nuclear RNA and lanes 4-6 contain cytoplasmic RNA.

Genomic DNA-free Cytoplasmic RNA:

RT-PCR was performed using human beta actin primers on cytoplasmic RNA isolated fom HeLa cells using Norgen's Cytoplasmic & Nuclear RNA Purification Kit. Lane M is the DNA ladder, lanes 1 and 3 are the negative control, lanes 2 and 4 show the expected 166 bp RT-PCR product. The lack of a product in lanes 1 and 3 indicate that no genomic DNA contamination is present in the isolated cytoplasmic RNA.

No Genomic DNA Contamination in Cytoplasmic Fraction:

Cytoplasmic and nuclear RNA isolated from HeLa cells using Norgen's kit was run on a 0.9% agarose 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).