nCounter Elements™ Fusion Genes
Multiplexed Fusion Gene Analysis
nCounter Elements can be used to create multiplexed assays capable of detecting and discriminating over 200 expressed gene fusions in a single reaction. Flexibility in design options and multiplexing capabilities enable development of assays for a variety of applications from discovery to diagnostics.
- Identify fusion events without knowledge of partner genes
- Characterize specific fusions by probing the junction sequence
- Characterize over 200 fusions in a single assay
- Study fusions and gene expression targets in the same assay
Universal Junction Sequence Designs
Targeting the Junction Sequence to Identify Specific Fusions
Highly specific probes for the junction sequence can be used to detect and discriminate unique fusion events. Junction probes can be combined with 5'/3' probes to create robust assays with orthogonal detection methodologies. Universal Junction Sequence Designs utilize toehold exchange technology to enable highly specific detection (Zhang 20121). View the tab below for more information on Universal Junction Sequence Designs.
5'/3' Imbalance Designs
Detect fusions without knowledge of the partner gene
Fusion events can be detected with a probe design that compares the ratio of gene expression upstream and downstream of the fusion junction. Each fusion partner may have promoters with different strength or activity, which will differentially affect expression of exons located 5' or 3' of this junction. A ratio of 5'/3' expression that diverges from 1 is therefore indicative that a fusion event has occurred. The 5'/3' design can be used to discover new fusions (Suehara 20122) or to develop robust assays to support clinical research programs (Lira 20133, Lira 20144).
Diagram of a 5'/3' imbalance design with 4 probes upstream and 4 probes downstream of the fusion junction. The number of probes can be varied depending on user preferences. View the tab below for more information on 5'/3' Imbalance Designs.
Fusion Gene Analysis
- Junction Sequence
- 5'/3' Imbalance
- Ordering Information
Multiplexed Analysis of BCR/ABL Fusions
A multiplexed nCounter Elements assay containing probes to seven distinct BCR-ABL fusions was run on total RNA from two cell lines, SUP-B15 and K562, each known to contain a specific fusion. Human Reference RNA was run as a negative control. Robust counts were obtained only for the correct fusion in each cell line; no crosshybridization was detected for any of the incorrect probes. The difference in signal for the two fusions reflects the difference in the expression levels of the fusion genes in the two cell lines.
Detect Fusions in the Presence of 90% Normal RNA
Unique Fusion Transcripts are detected in 50ng of total sample containing only 10% (5ng) fusion sample. RNA extracted from fusion-containing cell lines was mixed with Human Reference RNA in a 1:10 ratio. 50 ng of the combined RNA was run in a multiplexed nCounter Elements fusion assay. In each case the correct fusion was clearly detected. Cross-hybridization rates of all other fusion probes from the same fusion families were below background (data not shown). The differences in signal for the fusions reflect the differences in expression levels of the fusion genes in the cell lines.
5'/3' Imbalance Designs
A 5'/3' imbalance design enables detection of fusion events involving the ALK gene without knowledge of the fusion partner. For this assay, 4 probes were placed upstream and 4 probes downstream of the fusion junction. The data show the counts generated by these probes for an ALK wild type and an ALK fusion sample. In the sample containing an ALK fusion, there is a clear imbalance in the expression levels of the 5' probes compared to the 3' probes indicating that a fusion event has occurred. Data kindly provided by Kindstar Global.
|Product Type||Product Number||Description|
nCounter Elements GPR TagSet
General Purpose Reagents for 12 reactions; includes ERCCs
nCounter Elements GPR Extension TagSet
General Purpose Reagents for 12 reactions; no ERCCs
|nCounter Elements GPR Master Kit||
nCounter Elements GPR TagSets
A TagSet is a pool of nCounter Elements Reporter Tags and the Universal Capture Tag. Each Reporter Tag has a unique molecular barcode and tag that hybridizes to its complimentary sequence on target-specific oligonucleotide probes. The Universal Capture Tag enables the hybridized complex to be immobilized for counting. TagSets are available for analysis of 12 to 216 targets. Core TagSets are pre-mixed with ERCCs to enable analysis of 12 to 192 targets. Extension TagSets, without ERCCs, can be added to any core TagSet to expand the multiplexing capability by 12 or 24 targets.
nCounter Elements GPR Master Kits
Master Kits contain consumables and reagents for post-hybridization processing of samples. They facilitate magnetic bead purification to remove un-hybridized capture and reporter tags and also enable capture and immobilization of barcodes.
- Zhang et al. (2012) Optimizing the specificity of nucleic acid hybridization. Nat Chem 4(3):208-214.
- Suehara et al. (2012) Identification of KIF5B-RET and GOPC-ROS1 fusions in lung adenocarcinomas through a comprehensive mRNA-based screen for tyrosine kinase fusions. Clin Cancer Res 18(24):6599-6608.
- Lira et al. (2013) Multiplexed gene expression and fusion transcript analysis to detect ALK fusions in lung cancer. J Mol Diagn 15(1):51-61.
- Lira et al. (2014) A single-tube multiplexed assay for detecting ALK, ROS1, and RET fusions in lung cancer. J Mol Diagn 16(2):229-243.