RNA Subcellular Isolation Kit
isolate nuclear, cytoplasmic or total RNA
The RNA Subcellular Isolation Kit provides an efficient method to isolate nuclear and cytoplasmic RNA without cross-contamination. This method can be used to isolate both short and long RNA sequences (e.g. miRNA, piRNA, snoRNA, mRNA, and lncRNA). RNA molecules exist at different levels of maturation and processing within a cell at any given time. Such heterogeneity can bias downstream analysis and interpretation of data. By isolating RNA from specific subcellular fractions, the accuracy of gene expression analysis can be greatly improved. Subcellular isolation enhances the ability to detect low abundance transcripts and aids in the ability to study RNA processing dynamics by reducing the background from other intronic or mature RNAs. Additionally, subcellular isolation helps to localize long non-coding RNAs (lncRNAs) to their site of action, which can provide important insights into their functional role.
The RNA Subcellular Isolation Kit contains enough reagents to perform 15 nuclear and 15 cytoplasmic purifications from 2-4 million cultured cells or 15 mg of tissue per reaction. Simply lyse your samples, then separate the nuclear and cytoplasmic RNA by centrifugation. Each RNA fraction is loaded onto individual spin columns and washed to remove any proteins. Finally, the purified RNA is eluted from the column and quantified. The assay can also be used to isolate total RNA.
To learn more about our RNA Subcellular Isolation Kit, click on the Description, Method or Contents tabs below. To view a manual or other related documents, click on the Documents tab below.
|RNA Subcellular Isolation Kit Manual|
|Gene Regulation Products & Services Brochure|
|Newsletter - October 2015|
- Separates nuclear and cytoplasmic RNA without cross-contamination
- Works with cultured cells or tissue
- Easy-to-use spin columns included for RNA purification
- Method avoids the use of phenolic compounds
- Isolates RNA between the sizes of 15-7,000 nucleotides (includes miRNA, piRNA, snoRNA, mRNA, lncRNA)
- Purified RNA validated for use in RT-qPCR and RNA-seq
Figure 1: Agarose gel of nuclear and cytoplasmic RNA fractions.
Figure 2: TapeStation trace of cytoplasmic RNA isolated with the RNA Subcellular Isolation Kit.
Figure 3: Small RNA distribution frequency between cytoplasmic and nuclear fractions.
The RNA Subcellular Isolation Kit is designed to isolate nuclear and cytoplasmic RNA from cultured cells and fresh tissue, or total RNA from cultured cells and fresh or frozen tissue. The purified RNA is suitable for use in a variety of downstream applications.
- Reverse-transcription quantitative PCR (RT-qPCR)
- Northern blotting
- Purified RNA can undergo DNase I treatment
- Purified RNA can undergo ribosomal RNA (rRNA) depletion
- Purified RNA can be used for polyadenylation (poly A) selection
Figure 4: Subcellular RNA Isolation to identify RNA localization.
Figure 5: RNA-Seq data confirms lncRNA localization.
How does it work?
The RNA Subcellular Isolation assay works by lysing cell or tissue samples in complete lysis buffer. The lysate is then separated by centrifugation, with the supernatant containing cytoplasmic RNA and the pellet containing nuclear RNA. A guanidine-based buffer and ethanol are added to each RNA fraction before being loaded onto individual spin columns. Following a wash step to remove any proteins, the RNA fractions are eluted and quantified. RNA can be further process with Active Motif's DNase I Treatment Kit (Catalog No. 25502) to remove any genomic DNA contamination from the purified RNA sample prior to downstream analysis.
Figure 1: Flow Chart of the RNA Subcellular Isolation Method.
|Maximum Column Binding Capacity||40 µg|
|Maximum Column Loading Volume||750 µl|
|Size of purified RNA||15-7,000 nucleotides|
|Maximum Amount of Starting Material
2 x 106 cells (Total RNA)
4 x 106 cells (Cytoplasmic and Nuclear RNA)
15 mg tissue
|Assay Time||2 hours|
HeLa Cytoplasmic RNA
HeLa Nuclear RNA
HeLa Total RNA
12-25 µg at 250-500 ng/µl from 2 x106 cells
12-25 µg at 250-500 ng/µl from 2 x106 cells
25-40 µg at 500-900 ng/µl from 2 x106 cells
Contents & Storage
Please note that the RNA Subcellular Isolation Kits are shipped on dry ice and contain reagents with multiple storage temperatures inside. Please store each component at the temperature indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly.This kit includes the following components:
- 1 M DTT; Store at -20°C
- 10X PBS; Store at 4°C
- Lysis Buffer AM7; Store at 4°C
- Buffer G; Store at 4°C
- Purification Columns; Store at RT
- DEPC Water; Store at RT