Product Data Sheet
nCounter® Leukemia Fusion Gene Expression Assay Kit
nCounter®
Leukemia Fusion Gene Expression Assay
Product Highlights
• Simultaneous detection and quantification
of 25 fusion gene isoforms and 23
additional mRNAs related to leukemia
•Compatible with a variety of specimen
types, including FFPE, peripheral blood,
bone marrow, and cell lines
• 15 minutes hands-on time to analyze 12
samples and no specialized skills required
• Identify specific fusion gene isoforms for a
fraction of the cost of FISH or PCR
nCounter® Leukemia Fusion Gene Expression Assay Overview
The nCounter Leukemia Fusion Gene Expression Assay Kit allows researchers
to profile a comprehensive set of fusion genes which result from balanced
translocations in different leukemia sub-types. In addition to leukemia fusion
genes, the kit includes probes for 11 wild-type genes involved in the different
translocations, 12 leukemia-related biomarkers, and 2 normalization genes.
The 50-plex assay-kit facilitates cutting-edge research by leveraging the
advantages of the nCounter analysis system to provide robust analysis of a
variety of specimen types, including FFPE, with less hands-on time than any
other method available.
The nCounter Leukemia Fusion Gene Expression Assay Kit is offered for
research use only (RUO) and is ideal for characterization of retrospectively or
prospectively collected specimens, cell lines, and other sample types where a
comprehensive profile of fusion gene isoforms and leukemia biomarkers may
lead to important biological insights. Unique to the Leukemia Fusion Gene
Expression Assay is the Junction Probe design which enables detection of
fusion gene isoforms with high specificity. NanoString’s standard probe design
using “border probes” for the detection of fusion genes can only distinguish
between variants with different downstream exons. In order to differentiate
fusion genes with the same downstream exon, the leukemia fusion gene assay
utilizes Junction Probes which target the unique sequence spanning the fusion
junction of the two exons. In Figure 1, the design of the Junction Probes is
illustrated on the major and minor BCR-ABL fusion transcripts (Capture Probes
and Reporter Probes are denoted CP and RP respectively). NanoString has
optimized the probe design to achieve high sensitivity and specificity by
sequence modification.
Molecules That Count®
Translational Research
–
Gene Expression
–
miRNA Expression
–
Copy Number Variation
1
nCounter® Leukemia Fusion Gene Expression Assay Kit
Product Data Sheet
TEL-AML1 (e5e3)
RP-e1a2
PML-RARA (bcr2)
e1
CP-b2
b3
PML-RARA (bcr3)
MLL-AF4 (e11e4)
a2
MLL-AF4 (e11e5)
MLL-AF4 (e10e4)
RP-b2a2
b2
CP-b3
TEL-AML1 (e5e2)
MLL-AF4 (e10e5)
MLL-AF4 (e9e4)
a2
RP-b3a2
a2
Fusion Genes
CP-e1
BCR ABL
MLL-AF4 (e9e5)
MLL-AF4 (e8e7)
E2A-PBX1 (e13e2)
E2A-PBX1(e13-ins...
CBFß-MYH11 (e5e7)
CBFß-MYH11 (e5e8)
CBFß-MYH11 (e5e12)
Figure 1: Leukemia Junction Probes target the unique sequence spanning the fusion
junction.
AML-ETO
BCR-ABL (e1a3)
BCR-ABL (b2a3)
BCR-ABL (b3a3)
nCounter Leukemia Fusion Gene Expression
Assay Kit Performance
BCR-ABL (b3a2)
BCR-ABL (b2a2)
Translocation Partner Genes
To demonstrate the reproducibility of data generated via the nCounter Leukemia
Fusion Gene Assay, we processed total RNA isolated from bone marrow derived
from a leukemia patient prior to treatment. 500ng of total RNA was used per
(technical) replicate. Raw data was normalized to internal positive spike controls
present in each reaction to account for minor differences in hybridization and
purification efficiencies. Counts for individual probes in technical replicates
within a cartridge were highly correlated between sample preparations
(R2 > 0.99, Figure 3).
BCR-ABL (e1a2)
BCR
ABL1
AFF1 (AF4)
MLL
MYH11
CBFß
PBX1
E2A (TCF3)
ETV6 (TEL)
RARA
PML
TP53
RB1
Biomarker Genes
Each nCounter Leukemia Fusion Gene Assay generates 50 data points per
sample which characterize 25 fusion genes, 11 potential translocation partner
genes, 12 leukemia-related biomarkers, and 2 normalization genes. Figure 2
shows a complete data set for a single sample in which the assay identified the
correct fusion gene isoform and also generated additional information on the
expression levels of potential fusion partners and biomarkers.
BCR-ABL (e19a2)
NRAS
EVI1
MN1
ERG
MLLT11
PRAME
BAALC
FLT3
WT1
SOCS2
TBP
GUSB
0
5000
10000
Counts
Figure 2: A complete nCounter Leukemia Fusion Gene Profile shows the sample positive
for BCR-ABL b2a2 and provides additional information on gene expression for other
biomarkers and reference genes.
Figure 3: Counts for mRNA transcripts contained in the leukemia fusion gene assay in
total RNA from leukemic bone marrow were highly correlated between technical replicates.
2
As shown in Figure 1, the Junction Probe design allows for detection of fusion
gene isoforms in leukemia sub-types with high specificity. To demonstrate
the specificity of the Junction Probe design, the multiplexed probeset was
hybridized to individual synthetic RNA targets and then analyzed. Each probe
was evaluated based on the percentage of off-target counts that it generated.
Table 1 shows that all BCR-ABL isoform probes exhibited less than 1 percent
NanoString® Technologies
Product Data Sheet
cross-hybridization, indicating that the Junction Probe design for the nCounter
Leukemia Fusion Gene Expression Assay can accurately distinguish between
isoforms within a fusion gene family with great specificity. Similar experiments
were performed to verify less than 1 percent cross-hybridization between
isoforms for all other fusion gene families in the panel.
to positive controls included in the assay and typical background levels in these
assays are ~20 counts. Each fusion gene shown in the table was correctly called
by the nCounter Leukemia Fusion Gene Expression Assay. Biological variation
in expression levels of fusion genes is evidenced by the wide range of counts
seen for different fusion variants in samples derived from different patients. All
patient samples are from untreated individuals. The performance and workflow
advantages offered by the nCounter Leukemia Fusion Gene Expression Assay
Kit make it an ideal tool for advancing research in the area of leukemia fusion
gene variants. Visit our website to learn more (www.nanostring.com).
The nCounter Leukemia Fusion Gene Assay gives consistently reliable results
on RNA derived from a variety of sources. Table 2 shows data generated via the
nCounter Fusion Gene Expression Assay for different fusion gene isoforms from
total RNA isolated from a variety of sample types. Counts shown are normalized
Table 1: Low cross-hybridization between fusion genes of the BCR-ABL family demonstrates the high specificity of the junction probe design for the detection of specific isoforms.
BCR-ABL Targets
nCounter
Probes
e1a2
e1a3
b3a2
b2a2
b3a3
b2a3
e19a2
e1a2
100%
-
-
0.1%
-
-
-
e1a3
-
100%
-
-
-
-
-
b3a2
-
0.1%
100%
0.4%
-
-
-
b2a2
0.1%
-
-
100%
-
-
-
b3a3
-
-
-
-
100%
-
-
b2a3
-
-
-
-
0.1%
100%
-
e19a2
-
-
-
-
-
-
100%
Table 2: Counts generated for different fusion gene isoforms via the nCounter Leukemia
Fusion Gene Expression Assay in 500 ng of total RNA from various sample types (typical
background is ~20 counts).
nCounter Analysis System
The nCounter® Analysis System from NanoString offers a simple, cost-effective
way to profile hundreds of mRNAs, microRNAs, or CNVs simultaneously with
high sensitivity and precision. The digital detection of target molecules and
high levels of multiplexing eliminate the compromise between data quality and
data quantity, bringing better sensitivity, reproducibility, and linearity to your
results. It is ideal for studying defined gene sets across a large sample set, e.g.,
gene expression signature validation, pathway analysis, CNV validation studies,
and miRNA transcriptome analysis.
The system utilizes a novel digital technology that is based on direct multiplexed
measurement of gene expression and offers high levels of precision and
sensitivity. The technology uses molecular “barcodes” and single molecule
imaging to detect and count hundreds of unique transcripts in a single reaction.
Unlike other methods, the protocol does not include any amplification steps
that might introduce bias to the results.
After hybridization with the Leukemia Fusion Gene Assay CodeSet, samples
are transferred to the nCounter Prep Station where excess probes are removed
and probe / target complexes are aligned and immobilized in the nCounter
Cartridge. Cartridges are then placed on the nCounter Digital Analyzer for
data collection. Each mRNA transcript of interest is identified by the “barcode”
generated by six ordered fluorescent spots present on the Reporter Probe. The
Reporter Probes on the surface of the cartridge are then counted and tabulated
for each target.
Molecules That Count®
Sample Type
FFPE Bone Marrow
Fresh/Frozen
Bone Marrow
Fusion Gene
Isoform
BCR-ABL
b2a2
391
BCR-ABL
e1a2
1080
PML-RARA
bcr3
145
AML-ETO
e5e12
3439
BCR-ABL
b3a2
1080
BCR-ABL
b2a2
812
BCR-ABL
b3a3
988
BCR-ABL
e1a2
4587
1766
BCR-ABL
e1a3
CBFb-MYH11
e5e7
94
E2A-PBX1
e13e2
4762
PML-RARA
bcr2
535
PML-RARA
bcr1
502
PML-RARA
bcr3
1252
TEL-AML1
Peripheral Blood
Cell Lines
Counts
AML-ETO
e5e2
116
e5e12
5925
BCR-ABL
b3a2
1190
BCR-ABL
b2a2
2601
AML-ETO
e5e12
2292
BCR-ABL
b3a2
328
BCR-ABL
b3a2
16186
BCR-ABL
e1a2
370
BCR-ABL
b2a2
1962
E2A-PBX1
e13e2
5463
MLL-AF4
e9e5
1085
PML-RARA
bcr1
61
TEL-AML1
e5e2
237
3
nCounter® Leukemia Fusion Gene Expression Assay Kit
Product Data Sheet
nCounter Leukemia Fusion Gene Assay Target List
Fusion Genes
Disease
Translocation
Fusion Gene Family
Target
t(9;22)
BCR-ABL
b2a2
b3a2
b3a3
CML
b2a3
e1a3
e19a2
t(9;22)
BCR-ABL
e1a2
t(4;11)
MLL-AF4
e8e7
e9e5
e9e4
e10e5
e10e4
ALL
e11e5
e11e4
E2A-PBX1
e13-insertion-n27-e2
t(12;21)
TEL-AML1
e5e2
t(8;21)
AML-ETO
e5e12
Inv(16)
CBFß-MYH11
e5e12 (Type A)
t(1;19)
AML
e13e2
e5e3
e5e8 (Type D)
e5e7 (Type E)
t(15;17)
AML / APL
bcr1
PML-RARA
bcr3
bcr2
Translocation Partners
Fusion Gene Family
Biomarkers
Target
BCR-ABL
MLL-AF4
E2A-PBX1
SOCS2
ABL1
WT1
MLL
FLT3
AF4 (AFF1)
BAALC
E2A (TCF3)
PRAME
TEL (ETV6)
CBFß-MYH11
PML-RARA
Target
BCR
PBX1
TEL-AML1
Disease
AML, CML, ALL
MLLT11
ERG
CBFß
MN1
MYH11
EVI1
PML
NRAS
RARA
RB1
TP53
Normalization Genes
GUSB
TBP
4
Product Data Sheet
nCounter® Leukemia Fusion Gene Expression Assay Kit
Description
Specifications
Number of targets
25 Leukemia Fusion Genes
11 Translocation Partners
12 Leukemia - Biomarkers
Recommended amount of starting material
500ng purified total RNA
Sample types supported
RNA from a variety of sources including Bone Marrow, Peripheral Blood, FFPE
and Cell Lines*
Sample volume
5 μL
Hybridization reaction volume
30 μL
Limit of detection
≤ 0.5 fM
Spike titration linearity
R2 ≥ 0.95
Linear dynamic range
7 x 105 total counts
nCounter Prep Station throughput
12 samples < 2.5 hours
nCounter Digital Analyzer throughput
12 samples / 4 hours (up to 72 samples per day unattended running in continuous mode)
Controls
6 positive assay controls
8 negative assay controls
2 mRNA housekeeping controls
*Human samples should be derived from untreated individuals. Post-treatment individuals will likely have expression levels below the limit of detection.
Description
Quantity / Use
Part Number (P/N)
nCounter Leukemia Fusion Gene Expression Kit
12 assays
24 assays
48 assays
96 assays
FUS-LEU1-12
FUS-LEU1-24
FUS-LEU1-48
FUS-LEU1-96
nCounter Analysis System
(includes the Prep Station and Digital Analyzer)
1
NCT-SYS-120
Additional nCounter Prep Station
1
NCT-PREP-120
Additional nCounter Digital Analyzer
1
NCT-DIGA-120
NanoString Technologies, Inc.
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www.nanostring.com
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© 2012 NanoString Technologies, Inc. All rights reserved. NanoString®, NanoString Technologies®, nCounter®, Molecules That Count®, nSolver™, Plex2™, ChIP-String™ and miRGE™ are registered
trademarks or trademarks of NanoString Technologies, Inc., (“NanoString”) in the United States and/or other countries. All other trademarks and or service marks not owned by NanoString
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applications owned by NanoString or licensed to NanoString from Life Technologies Corporation and other third parties.
For research use only. Not for use in diagnostic procedures. v.20110718