Download nCounter® v2 Cancer CN Assay

Product Data Sheet
nCounter® v2 Cancer CN Assay
nCounter®
v2 Cancer CN Assay
Product Highlights
• Copy number analysis of 87 genes
commonly amplified or deleted in cancer
• Optimized for analysis of FFPE samples
• Unparalleled precision
• Accurate quantitation of highly amplified
genes
• Simple, robust assay protocol
nCounter® v2 Cancer CN Assay
The nCounter v2 Cancer CN Assay Kit is a highly multiplexed assay that enables copy number quantification for 87 genes commonly amplified or deleted in cancer
including PIK3CA, AKT, PTEN, BRCA, ERBB2, and MYC (TABLE 1). Based on NanoString’s proven digital, molecular counting chemistry, the assay delivers unparalleled
precision and accurately quantifies genes that are amplified to 10s or even 100s of copies. The assay is optimized for analysis of FFPE samples and shows excellent
correlation in comparisons of matched fresh frozen and FFPE samples.
For research use only, the nCounter v2 Cancer CN Assay Kit enables researchers to rapidly generate a copy number profile for hundreds of specimens per week. A
run can be set up with only 45 minutes hands-on time and results are available the next day (FIGURE 1).
Table 1: v2 Cancer CN Assay Gene List.
nCounter® Cancer CN Assay Gene List
AKT2
BCL2L2
CCNE1
CSMD1
ERBB2
GRB2
KIT
MDM2
MYCL1
PARK2
PTPRD
TERT
YAP1
AKT3
BIRC2
CDK4
DCC
FADD
HMGA2
KRAS
MDM4
MYCN
PAX9
RB1
TP53
YWHAZ
ZNF217
APC
BRCA1
CDK6
DCUN1D1
FGFR1
IGF1R
MAGI3
MELK
NCOA3
PDE4D
REG4
TP73
AR
BRCA2
CDKN1A
DYRK2
FHIT
IRS2
MAP2K4
MET
NF1
PDGFRA
REL
TRAF2
AURKA
C8orf4
CDKN2A
E2F3
FOXO1
ITGB4
MAP3K5
MITF
NKX2-1
PIK3CA
RPS6KB1
VEGFA
BBC3
CCND1
CDKN2C
EEF1A2
GAB2
JUN
MAPK7
MYB
NKX2-8
PRKCI
SHH
WHSC1L1
BCL2L1
CCND2
CRKL
EGFR
GPC5
KDR
MCL1
MYC
ORAOV1
PTEN
SKP2
WT1
Molecules That Count®
Translational Research
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Gene Expression
–
miRNA Expression
–
Epigenomics
–
Copy Number Variation
1
nCounter® v2 Cancer CN Assay
Product Data Sheet
The nCounter CN Assay protocol enables profiling in 4 easy steps:
45 Minutes Hands-on Time per Run
1
2
3
4
Fragment
Hybridize
Purify
Count
Fragment DNA either via Alul
digestion or Covaris AFA™.
Hybridize fragmented DNA with the
nCounter CodeSet.
Place hybridized samples in the
nCounter Prep Station for automated
post-hyb processing.
Transfer the nCounter cartridge from
the Prep Station to the nCounter
Digital Analyzer for image acquisition
and analysis.
30 minutes hands-on
5 minutes hands-on
5 minutes hands-on
5 minutes hands-on
Day 1
Day 1
Day 2 (automated)
Day 2 (automated)
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.
The system utilizes a novel digital technology that is based on direct multiplexed quantification of nucleic acids and offers high levels of precision and sensitivity. The
technology uses molecular “barcodes” and single molecule imaging to detect and count hundreds of unique targets in a single reaction. Unlike other methods, the
protocol does not include any amplification steps that might introduce bias to the results.
nCounter® CNV Assays
The nCounter CNV Assays allow researchers to quantify copy number for up to 800 regions of the human genome in a single multiplexed reaction. The nCounter
CNV Assays are based on the standard nCounter assay with two important additions: DNA fragmentation and denaturation. These two steps yield single-stranded
targets for hybridization with nCounter probe pairs which are comprised of a Reporter Probe which carries the signal, and a Capture Probe which allows the complex
to be immobilized for data collection. After hybridization, 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 in the nCounter Digital Analyzer for data collection. Each CNV probe
pair is identified by the “color code” 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.
Cancer CN CodeSet Design
The nCounter v2 Cancer CN CodeSet is designed to genes that have shown copy number variation in many cancers. Several publicly available sources were used to
generate the gene list (literature reviews, specific publications, public databases, feedback from experts). A preliminary list was generated combining genes from
all public sources and then prioritizing and selecting genes based on the frequency of membership in the source lists. The preliminary list of genes was then refined
further based on input from leading cancer researchers. The result is a list of 87 genes that are commonly amplified or deleted in cancer. The CodeSet includes
multiple probes per gene to increase accuracy when analyzing FFPE samples. Also included are 54 probes targeting invariant regions, bringing the total number of
probes in the CodeSet to 309.
2
0.0 MYCL1 1
2
3
Molecules That Count®
4
5
6
7
8
9
11
10
12
13
14
15
17
18
19
CRKL EEF1A2 MYCL1 CRKL EEF1A2 AURKA ZNF217 NCOA3 BCL2L1 BBC3 AKT2 CCNE1 DCC ITGB4 GRB2 RPS6KB1 BRCA1 ERBB2 NF1 MAPK7 MAP2K4 IGF1R PAX9 NKX2-­‐8 NKX2-­‐1 BCL2L2 IRS2 GPC5 RB1 FOXO1 BRCA2 MDM2 DYRK2 HMGA2 CDK4 KRAS CCND2 BIRC2 YAP1 GAB2 FADD ORAOV1 CCND1 WT1 PTEN TRAF2 MELK CDKN2A PTPRD MYC YWHAZ C8orf4 FGFR1 WHSC1L1 CSMD1 SHH MET CDK6 EGFR PARK2 MAP3K5 MYB VEGFA CDKN1A E2F3 APC PDE4D SKP2 TERT KDR KIT PDGFRA DCUN1D1 PIK3CA PRKCI MITF FHIT REL MYCN AKT3 MDM4 MCL1 REG4 MAGI3 JUN CDKN2C CCND2 HMGA2 DYRK2 KRAS copy number BIRC2 Copy
Number
CDK4 Tumor
1
KRAS YAP1 CRKL EEF1A2 AURKA ZNF217 NCOA3 BCL2L1 BBC3 AKT2 CCNE1 DCC ITGB4 GRB2 RPS6KB1 BRCA1 ERBB2 NF1 MAPK7 MAP2K4 IGF1R PAX9 NKX2-­‐8 NKX2-­‐1 BCL2L2 IRS2 GPC5 RB1 FOXO1 BRCA2 MDM2 DYRK2 HMGA2 CDK4 KRAS CCND2 BIRC2 YAP1 GAB2 FADD ORAOV1 CCND1 WT1 PTEN TRAF2 MELK CDKN2A PTPRD MYC YWHAZ C8orf4 FGFR1 WHSC1L1 CSMD1 SHH MET CDK6 EGFR PARK2 MAP3K5 MYB VEGFA CDKN1A E2F3 APC PDE4D SKP2 TERT KDR KIT PDGFRA DCUN1D1 PIK3CA PRKCI MITF FHIT REL MYCN AKT3 MDM4 MCL1 REG4 MAGI3 JUN CDKN2C MYCL1 TP73 CRKL EEF1A2 ZNF217 CRKL AURKA BCL2L1 AURKA NCOA3 EEF1A2 NCOA3 AKT2 ZNF217 BBC3 BBC3 DCC CCNE1 BCL2L1 CCNE1 GRB2 ITGB4 AKT2 BRCA1 ITGB4 RPS6KB1 DCC RPS6KB1 NF1 ERBB2 GRB2 MAP2K4 ERBB2 MAPK7 BRCA1 MAPK7 PAX9 IGF1R NF1 NKX2-­‐1 IGF1R NKX2-­‐8 MAP2K4 NKX2-­‐8 IRS2 BCL2L2 PAX9 BCL2L2 RB1 NKX2-­‐1 GPC5 BRCA2 GPC5 FOXO1 IRS2 DYRK2 FOXO1 MDM2 RB1 MDM2 CDK4 HMGA2 BRCA2 FF AURKA ZNF217 NCOA3 BCL2L1 BBC3 AKT2 CCNE1 DCC ITGB4 GRB2 RPS6KB1 BRCA1 ERBB2 NF1 MAPK7 MAP2K4 IGF1R PAX9 NKX2-­‐8 NKX2-­‐1 BCL2L2 IRS2 GPC5 RB1 FOXO1 BRCA2 MDM2 DYRK2 HMGA2 CDK4 KRAS CCND2 BIRC2 YAP1 GAB2 FADD ORAOV1 CCND1 WT1 PTEN TRAF2 MELK CDKN2A PTPRD MYC YWHAZ C8orf4 FGFR1 WHSC1L1 CSMD1 SHH MET CDK6 EGFR PARK2 MAP3K5 MYB VEGFA CDKN1A E2F3 APC PDE4D SKP2 TERT KDR KIT PDGFRA DCUN1D1 PIK3CA PRKCI MITF FHIT REL MYCN AKT3 MDM4 MCL1 REG4 MAGI3 JUN TP73 45.0 40.0 35.0 30.0 25.0 20.0 15.0 10.0 5.0 0.0 CDKN2C Number
Copy Copy
Number Tumor 2
5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 TP73 Copy Number
Tumor 3
FADD BIRC2 GAB2 CCND2 NanoString® Technologies
Product Data Sheet
Assay Performance
To evaluate the performance of the nCounter v2 Cancer CN Assay Kit in relevant sample types, we used the kit to characterize matching flash frozen and FFPE samples
from tumors. Data from three representative matched pairs are shown in Figure 2. The copy number profiles were highly concordant between the matching FFPE
and FF samples for both tumors. Several genes were clearly identified as deleted (see CDKN2C and CDKN2A for Tumor 1) or highly amplified (e.g., ERBB2, NCOA3,
ZNF217 for tumor 2). In addition, these data highlight the utility of the nCounter assay in identifying single copy deletions or amplifications, with several genes
showing either 1 copy (E2F3, MAP3K5, and BIRC3 ) or 3 copies (GPC5, IRS2, and NCOA3) in both FF and FFPE.
Correlation of Copy Number Calls for Matching Flash Frozen and FFPE Samples
Figure 2: Copy number for genes included in the Cancer CN Assay CodeSet for 3 matched pairs of FF and FFPE tumor samples. The data was analyzed using diploid reference samples
that matched the sample type as closely as possible. For the fresh frozen tissue, we used the cell line NA10851 as a control and for the FFPE samples we used the Cancer CN FFPE
Reference data set (provided by NanoString with the Cancer CN Assay kit). DNA was fragmented by AluI digestion and the input amount was 300ng.
FF FFPE FFPE FF FFPE Gene ID ID
Gene
FF FFPE Gene ID ID
Gene
5.0 4.0 4.5 3.0 3.5 2.0 2.5 1.5 Series1
1.0 Series2
0.5 Gene ID
20
22
Chromosome #
3
Product Data Sheet
nCounter® v2 Cancer CN Assay
Description
Specifications
Genomic regions analyzed in one reaction
87 genes commonly amplified or deleted in cancer (average 3 probes/region)
Recommended amount of starting material
300ng total genomic DNA
Sample types supported
Human genomic DNA from FFPE samples, fresh or frozen tissue, or cell lines
Reproducibility
> 99%
Number of copies detected
0 – 4, multiallelic
nCounter Prep Station throughput
12 samples < 2.5 hours
nCounter Digital Analyzer throughput
12 samples / 2.7 hours
Controls
54 invariant genomic regions, and spike-in process controls
Description
Quantity / Use
Part Number (P/N)
nCounter v2 Cancer CN Assay Kit
12 assays
24 assays
48 assays
96 assays
CNV-CAN2-12
CNV-CAN2-24
CNV-CAN2-48
CNV-CAN2-96
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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For research use only. Not for use in diagnostic procedures. v.20130321