nCounter
Analysis System®

Grant Application Package

NanoString Technologies, Inc. approves the use of images, graphs/charts, methods, and descriptions found in this grant package with proper citing
for grant proposal purposes. Please cite: [Image, data, methods, description] courtesy of NanoString Technologies, IncPlatform Overview

Technology Overview

The nCounter® Analysis System enables the profiling of 800+

NanoString’s nCounter technology is based on direct detection

mRNAs, microRNAs (miRNAs), and CNVs on one platformof oligonucleotides using color-coded molecular barcodeswith high sensitivity and precision. The primary benefits of thewhich are then used to individually count each of the target

platform are simplicity, reliability, and security. The workflow

molecules. The probe pair consists of a Reporter Probe, which

is simple, taking just 15 minutes of hands-on time and is

carries a fluorescent barcode on its 5’ end, and a Capture Probemostly automated. Direct, digital counting of individual target

which carries a biotin on its 3’ end, and each pair of probes

molecules using the nCounter’s patented molecular barcoding

hybridizes to a 100 base pair contiguous region on each target

chemistry and single molecule imaging ensure a dynamic range

oligonucleotide. The barcodes consist of six different positionsof over 5 logs as well as highly reproducible results, eliminatingwith each position one of four colors, creating a diverse number

the need for technical replicates. The assay does not requireof different barcodes and allowing for multiplexing of 800+

any reverse transcription or amplification, eliminating sources

targets in a single reaction tube.

of potential error and bias and enhancing the reliability of theresults. An additional advantage of nCounter chemistry is thatthe barcodes hybridize over a contiguous region of only 100
base pairs and there are no enzymatic steps, meaning thatthe assay is highly tolerant of difficult sample types such as
FFPE and crude-cell lysates. The nCounter Pro system features
advanced cyber security features such as an updated operating
system (Windows 10 IoT), hard drive encryption (TPM 2.0),
and data encryption (AES 256 and SHA-256) that enable partsof a 21 CFR Part 11 environment. All nCounter instruments andreagents are provided by NanoString Technologies and there
are a wide variety of off-the-shelf panels and assays for human,
mouse, rat, and non-human primate that enable research inoncology, immunology, and neuroscience.

The nCounter ® Pro Analysis System consists of two separate
instruments: the Prep Station and the Digital Analyzer.
Purification and binding of the hybridized complexes is carriedout automatically on the nCounter Prep Station. Magnetic
beads derivatized with short nucleic acid sequences thatare complementary to the Capture Probe and the Reporter
Probes are used sequentially. First, the hybridization mixture isallowed to bind to the magnetic beads by the Capture Probe.
Wash steps are performed to remove excess Reporter Probes
as well as oligonucleotides that are not hybridized. Afterwashing, the Capture Probes and Target/Probe complexes
are eluted from the beads and are hybridized to magnetic
beads complementary to the Reporter Probe. Wash steps
are performed, and excess Capture Probes are washed away.
Finally, purified Target-Probe complexes are eluted off and areimmobilized on a streptavidin coated surface in flow cells (thecartridge) for imaging and counting. Imaging and counting
are performed on the Digital Analyzer. Digital images areprocessed, and the barcode counts are tabulated in a comma
separated value (CSV) format. NanoString’s patented molecularbarcodes allow for true, digital detection technology ofindividual target molecules.

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nCounter Analysis System Grant Application Package • APR 2022
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

1. Prepare Sample

RNA prep

2. Hybridize

Minimum
input mass:25ng

3. Pool & Load

4. Analyze/reportmRNA expression
miRNA expression
Fusion transcripts

RNA input hyb

Pool & Load into a single nCounter®
cartridge lane (12 lanes/cartridge)

Immobilize and countmolecular barcodesnCounter Instruments
nCounter Pro
The nCounter Pro Analysis System consists of two separate
instruments: the Prep Station and Digital Analyzer. The
Prep Station is the automated liquid handling component of
the nCounter Pro. It processes samples post-hybridization
to prepare them for imaging and counting on the Digital
Analyzer. Prior to placing samples on the Prep Station,
samples are hybridized according to the nCounter protocol.
On the deck of the Prep Station, hybridized samples are
purified and subsequently immobilized in the sample cartridgenCounter Pro Prep Station & Digital Analyzer

for imaging and counting. The Digital Analyzer collects databy taking images of the immobilized Reporter Probes in thesample cartridge with a CCD camera through a microscope
objective lens. Images are processed internally, and data
output files include the target identifier and count number
along with a comprehensive tally of internal controls thatallows each assay to be quantitative. The resulting data filecan be distributed using a variety of methods and is easily
integrated with commonly used data analysis and visualization
software packages.
nCounter SPRINT Profiler
The nCounter® SPRINT Profiler is an all-in-one benchtop
nCounter system that combines the function of the Prep
Station and the Digital Analyzer into one instrument.
Hybridized samples are loaded into a SPRINT cartridge, wherea series of microfluidic chambers purifies and immobilizes thefluorescent Reporter Probes and readies them for imaging.
Images are processed, and output files include gene names
along with associated count numbers.

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nCounter Analysis System Grant Application Package • APR 2022
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

nCounter SPRINT Profiler

nCounter Data Analysis

nCounter CodeSets & Consumables

The ROSALIND® PlatformnCounter CodeSets

ROSALIND is a cloud-based software platform for life sciencenCounter CodeSets are pools of color-coded molecular barcodes

research that enables scientists to analyze and interpretand capture probes designed for specific projects. In addition todifferential gene expression data without the need fortarget-specific probes, a comprehensive mix of control probes

bioinformatics or programming skills. The platform is always

are added to each CodeSet for quantification, QC, and data

up to date: ROSALIND operates in a browser, eliminating thenormalization. Custom CodeSets and fixed content panels areneed to download software updates continually. ROSALIND

available for analysis of mRNA, miRNA, lncRNA, and DNA.

makes analysis of nCounter data easy, with guided modules
for quality control, normalization, pathway analysis, cell
type profiling, differential expression, and gene set analysis.
ROSALIND is free of charge for nCounter customers; a freeaccount can be created at www.rosalind.bio/nanostring.

nCounter Instrument Consumables
The nCounter Master Kit provides all the consumables
and reagents you need to perform any nCounter assay
on the nCounter Pro System. With each Master Kit order,
you receive nCounter Cartridges, Prep Plates, and anSolver™ Analysis Software

Prep Pack, which includes racked tips and foil piercers,

nSolver Analysis Software is a free, on-premises integrated

12 sample strip tubes, strip tube caps, tube sheaths,

analysis software platform for storage, custom QC, and custom

cartridge well seals and hybridization buffer. Masternormalization of nCounter data. Generate highly customized

Kits are available in packs for 12, 48 or 192 samples.

exports, basic statistical outputs, and publication-quality figures
quickly and easily with no incremental cost or bioinformatics
expertise. nSolver output data, provided in the form of a simple
CSV file, is extremely easy to both share and store due to thesmall file size. nSolver data is compatible with many standard
third-party analysis programs including Ingenuity Pathway
Analysis, Partek Genomics Suite, BioDiscovery Nexus Copy
Number and Advaita iPathwayGuide. For deeper insights
into your data, nCounter Advanced Analysis is a free, wizardbased add-on to the nSolver Analysis Software based on
robust R statistics. The nSolver Advanced Analysis module
enables an enhanced examination of experimental trends,
identification of pathway-specific responses, and profiling ofcell populations in an intuitive and sharable HTML report.

Running samples on the nCounter SPRINT Profiler requires
two types of consumables: the nCounter SPRINT microfluidic
sample cartridge and the nCounter SPRINT Profiler Reagent
Pack. The nCounter SPRINT Reagent Pack contains thebulk running buffers and reagents necessary to process
192 samples (16 runs, each run for up to 12 samples).

Training and support
Upon purchase and acquisition of an nCounter system,
NanoString provides on-site installation and calibration by atrained field engineer. Once the instrument has been qualified, afield application scientist provides a comprehensive introduction
and training for the nCounter platform. Training covers routine
use of the instrument, designing experiments, and performing

Data Analysis Service (DAS)

data analysis. Additionally, the field application scientist

NanoString’s Data Analysis Service (DAS) provides researchers

will perform wet lab experiments on-site to ensure that thewith the opportunity to leverage NanoString’s team ofcustomer has hands-on experience operating the instrument

scientists and bioinformaticians to provide meaningful

and performing assays. Upon successful completion ofand insightful analysis of your data quickly. This is a highly

training, the customer can access additional support regarding

consultative process that includes a one-on-one review

instrumentation, consumables, and software from NanoString’sof results with an expert highly trained in analyzing data

website or by contacting the NanoString Tech Support Team.

produced by the nCounter platform. Also offered are highly
comprehensive, interactive reports for the PanCancer IO
360™ and Breast Cancer 360™ Panels. Proprietary signature
analysis for the TIS, PAM50 and LST gene signatures can be
added to any standard DAS project on an a la carte basis.

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nCounter Analysis System Grant Application Package • APR 2022
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

nCounter Sample Types & Supporting DatanCounter assays can be used with a wide variety of different sample types such as purified total RNA, raw cell or blood lysatesand formalin-fixed, paraffin-embedded (FFPE) tissue sections with no loss in precision. Even severely degraded RNA can be used,owing to the fact that the nCounter probe sets only hybridize to a 100 base pair contiguous region of the target oligonucleotide.
Gene Expression Profiling of mRNA
Gene counts from two technical replicates plotted against
one another demonstrate the assay reproducibility overa wide dynamic range (10-50,000 counts). One total
RNA sample was split into two separate hybridization
reactions and processed independently on the nCounter
Analysis System. In this experiment 75 counts are equal
to a concentration of approximately 1 copy per cell. Thisdata illustrates the high level of sensitivity and precisionof the assay even at very low expression levels.
miRNA
To demonstrate the reproducibility of the miRNA assay,
we used the nCounter Human miRNA Expression Assay
Kit to analyze commercially purchased human total
RNA. Replicate samples were prepared and processedon the instrument individually. When plotted against
one another, the samples demonstrated very highreproducibility, with R 2 values of greater than 0.99.
10,000Whole Blood Lysates
Gene expression counts from two PAXgene-lysed
whole blood replicates were compared to that from
100 ng of matched purified total RNA. Results using
blood lysates were highly correlated with purified RNA(R 2 > 0.96 and R 2 > 0.97) and demonstrated that highquality data can be obtained using PAXgene-lysed
whole blood. (PAXgene is a trademark of Qiagen).

5nCounter Analysis System Grant Application Package • APR 2022
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

10,000 cells
5,000 cells

Lysate Counts

1,000

2,500 cells

100
10
1

1

10,000

Lysate Counts

Crude Cell Lysates
Gene expression counts from three different cell lysate
preparations made of 2,500, 5,000, and 10,000 cells werecompared to those from 100 ng of purified total RNA. Results
using cell lysates were highly correlated with that frompurified RNA (R 2 > 0.97 for all three) and demonstrated thatcomparable data can be achieved with either protocol.

10100
Total RNA Counts

1,000

10,00010100
Total RNA Counts

1,000

10,000

Lysate 1
Lysate 2

1,000
100
10
1

1

Formalin-Fixed Paraffin-Embedded Tissue (FFPE)
Gene expression counts from RNA derived from FFPE werecompared to that from matched purified total RNA and theresults were highly correlated with purified RNA (R 2> 0.84),
demonstrating that high quality data can be achieved fromFFPE tissue sections using the nCounter Analysis System.

Analysis of miRNA from FFPE Samples
Formalin-Fixed, Paraffin-Embedded (FFPE) tissue sections are
difficult to analyze due to RNA degradation. To demonstrate
the ability of the miRNA assay to analyze degraded RNA
samples, we compared gene expression counts of miRNA
from purified total RNA from FFPE liver sections withthat from purified total RNA from matched fresh-frozen
tissue using the nCounter Human miRNA Expression Assay
Kit. Counts for all detected miRNA species were highly
correlated (R 2 > 0.95) between the two sample types.

Analysis of Degraded and Low RNA Input Samples
The nCounter Low RNA Input Kit enables high quality gene
expression profiling of up to 800 gene targets from as little
as 1 ng of sample. The kit is optimized for use with RNA fromFFPE tissue as well as crude cell lysates. Additionally, thekit can be utilized for the study of low expressing genes.
The streamlined, user-friendly workflow and reliable results
enable gene expression studies of low sample inputs or low
expressing genes to be completed quickly and efficiently.

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nCounter Analysis System Grant Application Package • APR 2022
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

Fold Change Low RNA Input (1 ng) UHR/HBR

Analysis of DNA Purified from FFPE Samples
This figure shows copy number counts for genes includedin the nCounter Cancer CN Assay for two matched pairs ofFresh Frozen (FF) and FFPE tumor samples. The data wasanalyzed using diploid reference samples matching the sample
type as closely as possible. For the fresh frozen tissue, weused 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 wasfragmented by AluI digestion using an input amount of 300 ng.

5
4
3
2
1
-5

1

-3

0
-1
-1
-2
-3
-4
-5

1

3

Fold Change Standard assay (100 ng) UHR/HRB

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Justification for Using Nanostring technology
to Accelerate Your ResearchHigh Reproducibility
The nCounter system is known for its high level of
reproducibility and precision, as has been shown in various
studies by independent research groups. Veldman-Joneset al. (2015) reported correlation scores >0.98 between
technical replicates, irrespective of whether the RNA wasderived from fresh cells or FFPE material. Similarly, Balkoet al. (2012) investigated systematically the consistency
between repeated nCounter measurements from breast
cancer FFPE samples and found consistent R 2 values >0.99(Supplemental material). In another study investigating
molecular subgrouping of medulloblastoma, researchers
looked at inter-operator variance and concordance between
results obtained from the same sample sets at different
processing sites and reported excellent correlation scores>0.97 (Northcott et al., 2012). nCounter panels include
numerous controls that serve as indicators for technicalvariability present in the measurement of a particular sample.
High Level of Robustness, Even with Poor Quality Samples
There are many publications that clearly demonstrate gene expression analysis can be done reliably and accurately on FFPE
material using the nCounter Analysis System, often where highfailure rates are observed using RNASeq with the same material (Omolo et al., 2016; Lesluyes et al., 2016; Le Guellec et al.,2018). Sample quality requirements (i.e., RNA integrity numbers) are much higher for sequencing assays which can also
be negatively affected by background contaminants, including
chemical fixatives used in the FFPE process. In contrast, theshort sequence (100 bp) required for nCounter probe detection
and the fact that library preparation is not necessary results in
a considerably better tolerance of the nCounter assay towards
RNA quality. For example, Lesluyes et al. (2016) failed to reliably transfer an RNASeq-based signature for prediction of metastatic outcome in sarcomas from fresh frozen samples to FFPE
tissue, as it was not possible to obtain sequencing results from
more than 50% of the FFPE samples. Le Guellec et al. (2018)confirmed this experience in their study, where successful measurement of a particular signature using RNASeq occurred inonly 17 out of 67 FFPE samples (25%), whereas all 67 samples(100%) were analyzed using the nCounter Analysis System.

Technology offers higher sensitivity than targeted RNASeq
While the correlation of expression level measurements between RNASeq and the nCounter platform is typically quite
good overall (Leong et al., 2015), the concordance remains
even for genes with low expression levels. Steijger et al. (2013)
showed that the discrepancy is usually associated with small,short read numbers representing low abundant targets in RNASeq data sets and the failure of downstream analysis packages
to robustly infer presence and expression of these genes. In
contrast, counts for such low abundance targets are clearly removed from background noise levels when looking at nCounter
data, demonstrating a higher sensitivity of the platform.
Faster Processing time
The total time from input sample to results on the nCounterplatform is less than two days. In contrast, the amount oftime to perform library preparation, QC/titration, and sequencing typically exceeds four days for RNASeq-based
assays. The difference in actual hands-on time is even more
pronounced, with 45 mins or less (only 15 minutes for abasic gene expression assay) required for the nCounterversus four hours or more for the average RNASeq run.
Simple Data Analysis
Data analysis of nCounter experiments can be performed
easily. Data files are small and can be processed withoutsophisticated software tools, eliminating the need for extensive computing infrastructure or highly specialized bioinformaticians. NanoString offers an openly available, on-premises
analysis software package (nSolver) for PC and Mac thatfurther streamlines the QC and analysis steps. In addition,
the ROSALIND Platform is available for intuitive data analysis
in the cloud that can be easily shared and collaborated on.
Truly orthogonal validation technology
Because nCounter assays do not require reverse transcription, target amplification or library preparation, thenCounter is particularly well suited for orthogonal validationof candidate genes identified by sequencing, PCR, or array-based methods. An increasing number of studies employ
the nCounter Analysis System for target validation (Sun etal., 2011; Smallridge et al., 2013; Bhargava et al., 2013; Sabo
et al., 2014; Owonikoko et al., 2014; Graw et al. 2015).

Publications
The utility of the nCounter system is demonstrated in thousands of peer-reviewed publications, many in top journals.
Visit www.nanostring.com/resources/publications to review the latest nCounter publications.

For more information, please visit nanostring.com
NanoString Technologies, Inc.
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FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
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