Cytoplasmic & Nuclear RNA Purification Kit
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. Norgens 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 Norgens 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.
Norgens 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. |