nCounter Elements™
General Purpose Reagents
PACKAGE INSERT
NanoString Technologies, Inc.
530 Fairview Ave N
Suite 2000
Seattle, Washington 98109
www.nanostring.com
Tel:
206.378.6266
888.358.6266
E-mail: [email protected]
Molecules That Count®
Gene Expression
–
Copy Number Variation
–
Single Cell Gene Expression
LBL-C0266-03
nCounter Elements™ General Purpose Reagents
PACKAGE INSERT
FOR LABORATORY USE.
Intellectual Property Rights
This nCounter Elements™ manual and its contents are the property of NanoString Technologies, Inc. (“NanoString”), and is intended solely
for the use of NanoString customers, for the purpose of using nCounter Elements™ General Purpose Reagents. nCounter Elements™ and this
User Guide and any other documentation provided to you by NanoString in connection therewith, are subject to patents, pending patents,
copyright, trade secret rights, and other intellectual property rights owned by, or licensed to, NanoString.
Trademarks
NanoString®, NanoString Technologies®, nCounter®, Molecules That Count®, nDesign™, and nCounter Elements™ 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 that appear in this document are the property of their respective owners.
Copyright
© 2013 NanoString Technologies, Inc. All rights reserved.
Storage Conditions (TagSets)
Document Information
Version 03, created 2013-12
LBL-C0266-03
2
NanoString® Technologies
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PREFACE............................................................................................................................................................................................ 4
Conventions Used............................................................................................................................................................................. 4
Note Types........................................................................................................................................................................................... 4
Fonts...................................................................................................................................................................................... 4
Procedures........................................................................................................................................................................................... 4
Contact Information........................................................................................................................................................................ 4
CHAPTER 1: Introduction...................................................................................................................................................... 5–8
Overview............................................................................................................................................................................................... 5
nCounter Elements Technology................................................................................................................................................. 5
Designing Oligonucleotide Probes with nDesign Gateway.......................................................................................... 6
CodeSet Design................................................................................................................................................................ 6
Materials Provided............................................................................................................................................................................ 8
Contents Description...................................................................................................................................................... 8
Storage and Handling.................................................................................................................................................... 8
Suggested Materials........................................................................................................................................................................ 8
CHAPTER 2: RNA Hybridization Guidelines................................................................................................................ 9-11
Overview............................................................................................................................................................................................... 9
Setting Up 12 Hybridization Reactions for RNA................................................................................................................. 9
CHAPTER 3: DNA Hybridization Guidelines..............................................................................................................12-15
Overview..............................................................................................................................................................................................12
Fragmentation..................................................................................................................................................................12
CNV Reference Sample Considerations................................................................................................................12
Denaturation.....................................................................................................................................................................13
DNA Input Amount........................................................................................................................................................13
DNA Quality.......................................................................................................................................................................13
Setting Up 12 Hybridization Reactions for DNA................................................................................................................14
CHAPTER 4: Creating Oligonucleotide Probe Pools........................................................................................... 16-18
Overview..............................................................................................................................................................................................16
Creating Master Probe Stocks...................................................................................................................................................17
Creating 30X Working Probe Pools........................................................................................................................................18
CHAPTER 5: Evaluating Performance.........................................................................................................................19-23
Technical Replicates.......................................................................................................................................................................19
Sample Input Titration................................................................................................................................................................. 20
Qualifying Probes to Confirm Low Background...............................................................................................................21
Calculating Correction Factors between Lots...................................................................................................................23
SYMBOLS AND DEFINITIONS................................................................................................................................................24
Molecules That Count®
Gene Expression
–
Copy Number Variation
–
Single Cell Gene Expression
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nCounter Elements™ General Purpose Reagents
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Conventions Used
The following conventions are used throughout this manual and are described below for your reference:
Note Types
Special font formatting is used in this manual. Such formatting conventions are used in specific instances as described below:
NOTE
This note type emphasizes general information.
IMPORTANT
This note type presents essential content indicating that the potential exists for assay failure, diminished
data quality, and/or a loss of data if the information presented is ignored.
BOLD When appearing in text or in a procedure, the bold text serves to highlight a specific action or refer to another step in the instruction manual.
• Bold text is also used to highlight important text or terms.
• Blue text is used to help the reader identify active hyperlinks.
ITALICS
Used to emphasize an important word or expression within the text.
• Formatting of a book title, journal, or other documentation.
• Used to indicate the special or unusual meaning of a word or phrase.
Procedures
Numbered procedures appear frequently providing step-by-step instruction for accomplishing a task. Typically, a numbered step provides
direction for a specific action and may be followed by the expected response. Additional information may be presented in the form of a
specific note type, bullets, or image important to facilitate clarity and understanding.
NanoString Technologies, Inc.
530 Fairview Ave N
Suite 2000
Seattle, Washington 98109 USA
4
Tel:
206.378.6266
888.358.NANO (6266)
Fax:
206.378.6288
Email:
[email protected]
PACKAGE INSERT
1
Introduction
Overview
This manual describes the recommended procedures for setting up hybridization reactions utilizing nCounter Elements™ General Purpose
Reagents (GPRs). nCounter Elements are flexible, off-the-shelf reagents that can be used to develop assays for measuring digital counts of
nucleic acid targets. Assays for gene expression can be performed using samples in the form of cell or tissue lysate or total RNA. Total RNA
can be isolated from a variety of biological samples including blood, tissue, and Formalin-Fixed Paraffin-Embedded (FFPE) tissue. Assays to
detect DNA, such as copy number variation, can also be performed (fragmentation and denaturation of isolated DNA is necessary for efficient
hybridization).
All nCounter Elements reagents are based on NanoString’s core technology for measuring the abundance of nucleic acids via digital detection
of fluorescent molecular barcodes. Products described in this manual allow for detection of up to 216 unique target sequences.
This manual describes in detail the recommended methods for setting up nCounter Elements hybridization reactions when working with
RNA or DNA as well as considerations for ordering target-specific oligonucleotide probes. If planning to continue with post-hybridization
processing and data analysis using the nCounter® Prep Station or the nCounter Digital Analyzer, please review detailed instructions in the
appropriate manuals.
nCounter Elements Technology
NanoString’s nCounter Elements technology is based on molecular barcoding and digital quantification of target RNA or DNA sequences
through the use of an nCounter Elements TagSet and target-specific oligonucleotide probe pairs (supplied by the user). The TagSet consists
of fluorescently labeled specific Reporter Tags and a biotinylated universal Capture Tag (Figures 1.1 and 1.2). The Reporter Tags each have a
unique pattern of six spots of color, creating fluorescent barcodes that can be individually resolved and counted during data collection. The
universal Capture Tag anchors each target to the surface of a streptavidin-modified surface, such as a glass cartridge, during this process.
Barcode 1
Tag T001
Barcode 2
Tag T002
Barcode 3
Tag T003
FIGURE 1.1: Examples of Reporter Tags with unique fluorescent barcodes and recognition sequences.
Molecules That Count®
Gene Expression
–
Copy Number Variation
–
Single Cell Gene Expression
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nCounter Elements™ General Purpose Reagents
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During hybridization, the specific Reporter Tags and universal Capture Tag hybridize to a pair of target-specific oligonucleotide probes,
which in turn hybridize directly to the single-stranded RNA or DNA target (Figure 1.2). Probe A hybridizes to a specific Reporter Tag and
the 5’ region of the target nucleic acid sequence. Probe B hybridizes to the universal Capture Tag and the 3’ region of the target nucleic acid
sequence. Each complete structure—containing the target RNA or DNA, two oligonucleotide probes, and the Reporter and Capture Tags—is
referred to as a Tag Complex.
Reporter Tag
A unique tag sequence
is assigned to each
target sequence
Universal Capture Tag
Probe A**
¦
¦
¦
5’
Probe B**
3’
Nucleic Acid Target
FIGURE 1.2: Customized oligonucleotide probes hybridize with general purpose Reporter and Capture Tags and the target nucleic acid to create a Tag
Complex.
The TagSet and probes are present in the reaction in large excess to the target molecules to ensure that each target undergoes hybridization.
After hybridization, excess probes and tags as well as non-target nucleic acids may be washed away using a two-step magnetic bead-based
purification process.
Magnetic beads derivatized with short nucleic acid sequences that are complementary to either the universal Capture Tag or the Reporter
Tags may be used sequentially. First, the hybridization mixture is allowed to bind to magnetic beads that interact with the Capture Tag. Wash
steps can then be performed to remove excess Reporter Tags and probes as well as non-target nucleic acids. After washing, complexes
can be eluted off the beads and hybridized to a second set of beads complementary to the Reporter Tags. Additional wash steps may be
performed to remove excess Capture Tags and probes. Finally, the purified Tag Complexes can be eluted off the beads and immobilized for
data collection.
NOTE:Purification and immobilization of Tag Complexes can be automated using the nCounter Prep Station. Data collection can be
automated using the nCounter Digital Analyzer. Please review detailed instructions in the appropriate manuals.
Designing Nucleotide Probes with nDesign Gateway
Prior to sample hybridization, oligonucleotide probes should be ordered separately for each target RNA or DNA. The oligonucleotide probes
should contain both tag- and target-specific sequences that effectively link each target to a specific Reporter Tag and to the universal
Capture Tag. Because Reporter and Capture Tags do not directly hybridize with the targets of interest, generating new oligonucleotide
probes enables a common TagSet to be used to label and detect many different RNA or DNA targets.
NanoString provides an online tool, nDesign Gateway, which enables users to design probes for gene expression assays. Users may select
specific probe sequences for human, mouse, and rat genes of interest and then establish associations between targets and Reporter Tags.
Design data created using nDesign Gateway can be downloaded and submitted to a third-party oligonucleotide manufacturer.
Please see the instructions provided on the nDesign Gateway home page for additional information on the use of this tool. Alternatively,
contact customer support ([email protected]) to discuss other options and tools available to facilitate CodeSet design.
CodeSet Design
The nCounter Elements CodeSet is comprised of the TagSet and the corresponding oligonucleotide probes (supplied by the user) that
recognize the targets of interest (Figure 1.3). Sixteen different core TagSets are available in increments of 12 tags. It is important to remember
that larger TagSets include all tags found in smaller TagSets. For example, a core TagSet of 12 tags includes T001 through T012, and a core
TagSet of 24 tags includes T001 through T024. The largest core TagSet currently available can detect up to 192 targets.
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NanoString® Technologies
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Reporter Tag
Probe A
Capture Tag
Probe B
FIGURE 1.3: Combining Elements TagSet
general purpose reagents with user-supplied
oligonucleotide probes produces a custom
Elements CodeSet for the detection of a
specific set of targets.
CodeSet
Two core TagSets cannot be combined to increase the number of targets in an experiment as some of their Reporter Tags will overlap. To add
additional targets to an ongoing experiment, use an Extension TagSet, which is available for 12 or 24 targets and does not include controls.
The unique Reporter Tags in the Extension TagSets do not coincide with any of those found in the core TagSets.
Assigning reference genes and other frequently analyzed targets to Reporter Tags with the lowest tag number will make the most efficient
use of assay reagents in experiments where the target set is expected to evolve over the course of the project. In this way the same
oligonucleotide probes can be used for common targets in more than one experiment. There are multiple ways to examine additional targets
in an assay: (1) order a larger TagSet, (2) order an Extension TagSet, or (3) order new oligonucleotide probes that link Reporter Tags in the
existing TagSet with new targets.
Core TagSet
Core TagSet
Core TagSet
Extension TagSet
Tag
Target
Tag
Target
Tag
Target
Tag
Target
T001
Gene A
T001
Gene A
T001
Gene A
X001
Gene M
T002
Gene B
T002
Gene B
T002
Gene B
X002
Gene N
T003
Gene C
T003
Gene M
T003
Gene C
X003
Gene O
T004
Gene D
T004
Gene N
T004
Gene D
X004
Gene P
T005
Gene E
T005
Gene E
T005
Gene E
X005
Gene Q
T006
Gene F
T006
Gene F
T006
Gene F
X006
Gene R
T007
Gene G
T007
Gene G
T007
Gene G
X007
Gene S
T008
Gene H
T008
Gene O
T008
Gene H
X008
Gene T
T009
Gene I
T009
Gene P
T009
Gene I
X009
Gene U
T010
Gene J
T010
Gene Q
T010
Gene J
X010
Gene V
T011
Gene K
T011
Gene R
T011
Gene K
X011
Gene W
T012
Gene L
T012
Gene L
T012
Gene L
X012
Gene X
FIGURE 1.4: An existing CodeSet (A) can be modified by reassigning Reporter Tags to new targets (B) or adding an extension TagSet (C). Targets that have
been added or replaced are indicated in blue.
nDesign Gateway stores information on all past orders and will recommend a probe/tag configuration that facilitates efficient re-use of
previously selected oligonucleotide probes. The Tag Reporter Probe Mapping page also provides information on probe/tag assignments and
enables users to make changes as desired.
Molecules That Count®
Gene Expression
–
Copy Number Variation
–
Single Cell Gene Expression
7
nCounter Elements™ General Purpose Reagents
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Materials Provided
TagSets are available for analysis of 12 to 216 targets. Core TagSets are pre-mixed with a comprehensive set of controls and enable analysis
of 12 to 192 targets depending on the product ordered. Extension TagSets, provided without controls, can be added to any core TagSet to
expand the multiplexing capability by 12 or 24 targets. Each nCounter Elements TagSet is provided with sufficient material to perform 12
hybridization reactions.
TABLE 1.1: Materials provided for nCounter Elements hybridization reactions
Product Type
Description
Product Number
nCounter Elements TagSet
(Contains reagents for 12 reactions; includes controls)
12 Tags
24 Tags
36 Tags
48 Tags
60 Tags
72 Tags
84 Tags
96 Tags
108 Tags
120 Tags
132 Tags
144 Tags
156 Tags
168 Tags
180 Tags
192 Tags
ELE-P1TS-012
ELE-P1TS-024
ELE-P1TS-036
ELE-P1TS-048
ELE-P1TS-060
ELE-P1TS-072
ELE-P1TS-084
ELE-P1TS-096
ELE-P1TS-108
ELE-P1TS-120
ELE-P1TS-132
ELE-P1TS-144
ELE-P1TS-156
ELE-P1TS-168
ELE-P1TS-180
ELE-P1TS-192
nCounter Elements Extension TagSet
(Contains reagents for 12 reactions; no controls provided)
12 Tags
24 Tags
ELE-P1EX-012
ELE-P1EX-024
Contents Description
nCounter Elements TagSet – 12 Reaction Aliquot (1 x 65 μL) buffer, nucleic acids, nucleic acids with fluorescent dyes.
nCounter Elements Extension TagSet – 12 Reaction Aliquot (1 x 70 μL) buffer, nucleic acids, nucleic acids with fluorescent dyes.
Storage and Handling
The expiration date for the nCounter Elements TagSet is listed on the outer box labeling. Reagents must be stored at -80°C.
TABLE 1.2: Suggested materials for nCounter Elements assays
8
Product Type
Description
Product Number
nCounter Elements Master Kit
(Master Kits contain the following components: Hybridization Buffer,
Prep Pack(s), Prep Plates, and Cartridges)
48 Reactions
192 Reactions
ELE-AKIT-048
ELE-AKIT-192
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2
RNA Hybridization Guidelines
Overview
nCounter Elements technology can be used to detect RNA for measuring gene expression. These reagents are compatible with a wide variety
of sample types, including purified total RNA and whole cell lysate as well as RNA purified from FFPE samples. See below for recommended
instructions for hybridizing the Reporter and Capture Tags, oligonucleotide probes, and RNA sample. If using a sample type that is not
addressed in this manual, please contact customer support for additional information ([email protected]).
IMPORTANT: Do not vortex or pipet vigorously as this may shear the Reporter Tags. Mix only by flicking or inverting tubes. A
picofuge is preferable when spinning down solutions due to its low speed. If using a microfuge, do not “pulse” the machine as
it may reach maximum speed and spin the TagSet out of solution. Spin at less than 1,000 rpm for no more than 30 seconds.
NOTE: The following guidelines cover general steps for setting up RNA hybridization reactions. Each specific application using nCounter
Elements GPRs will require optimization and validation using appropriate performance metrics defined by the end user.
All reagents are provided for multiples of 12 reactions. Scale up the suggested volumes for additional reactions. Note that the instructions for
12 reactions use a multiplier of 13 to allow for dead volume in the master mix.
Molecules That Count®
Gene Expression
–
Copy Number Variation
–
Single Cell Gene Expression
9
nCounter Elements™ General Purpose Reagents
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Each final hybridization reaction will have a volume of 30 μL and contain the following components: 10 μL of hybridization buffer, 5 μL of
TagSet, 1 μL of 30X Working Probe A Pool (0.6 nM each Probe A), 1 μL of 30X Working Probe B Pool (3 nM each Probe B), and up to 13 μL of
sample RNA. If using an Extension TagSet, add 5 μL of the Extension TagSet, 1 μL of 30X Extension Probe A Pool and 1 μL of 30X Extension
Probe B Pool and reduce the total volume of sample to no more than 6 μL (Figure 2.1). Instructions on preparing 30X Working Probe Pools
are provided in Chapter 4.
FIGURE 2.1: Guide to creating master mix and preparing RNA hybridization reactions.
Dilute Master Probe Stocks to 30X Working Probe Pools
30X Working Probe A Pool
30X Working Probe B Pool
TagSet
Hybridization Buffer
Aliquot master mix to each tube
Add sample RNA to each tube (see guidelines)
Add nuclease-free water, if needed, to reach a
final volume of 30 μL
Hybridization reactions at 67°C for at least 16 hours
Each Reaction
13 Reactions
Hybridization Buffer
10 μL
130 μL
TagSet
5 μL
65 μL
30X Working Probe A Pool
1 μL
13 μL
30X Working Probe B Pool
1 μL
13 μL
Sample*
Up to 13 μL
Total Volume
30 μL
Each Reaction
13 Reactions
Hybridization Buffer
10 μL
130 μL
TagSet
5 μL
65 μL
Extension TagSet
5 μL
65 μL
30X Working Probe A Pool
1 μL
13 μL
30X Extension Probe A Pool
1 μL
13 μL
30X Working Probe B Pool
1 μL
13 μL
30X Extension Probe B Pool
1 μL
13 μL
Sample*
Up to 6 μL
Total Volume
30 μL
*NanoString recommends preparing samples with a high concentration
to facilitate reducing the sample volume if an extension TagSet is later
necessary.
IMPORTANT: Master Probe Stocks must be diluted to 30X Working Probe Pools (see page 18). Addition of concentrated Master
Probe Stocks directly to the assay will result in assay failure.
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NanoString® Technologies
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1. Follow the directions in Chapter 4 to create the 30X Working Probe Pools. The accuracy of the probe concentration in the hybridization
reaction is important for maximizing sensitivity. Be sure to dilute the pools to the appropriate 30X concentration before addition to
the master mix below.
2. If the samples contain total RNA, go to Step 4.
3. If the samples will be cell or tissue lysates, prepare them according to the recommended instructions in your supplier’s kit (e.g., see
Qiagen® RNeasy™ Mini Handbook, supplied with Qiagen® product numbers 74104 and 74106).
a. Cells or tissues should be lysed at an appropriate concentration so that no more than 3 μL of lysis buffer is added to the final 30 μL
reaction. Larger amounts of lysis buffer will impact the melting temperature of the probes during hybridization and lead to lower
sensitivity. For reference, 10,000 mammalian cells yield approximately 100 ng of total RNA.
b. Lysates should be aliquoted and stored at -80°C. Avoid freeze/thaw cycles.
4. Remove an aliquot of the TagSet from the freezer and thaw it on ice. Invert several times to mix well, and briefly spin down the reagent
at less than 1,000 rpm.
5. Each core TagSet tube contains 65 μL of reagent. Create a master mix by adding reagents directly into the TagSet tube. First add
130 μL of hybridization buffer, followed by 13 μL of the 30X Working Probe A Pool. Mix well by flicking the tube and briefly spin down
at less than 1,000 rpm before adding 13 μL of the 30X Working Probe B Pool. Mix and spin again.
a. If using an Extension TagSet, also add 65 μL of the Extension TagSet reagent and 13 μL of the 30X Extension Probe A Pool before
mixing. Then also add 13 μL of the 30X Extension Probe B Pool before the final mixing step.
b. If all samples have the same volume, additional nuclease-free water may be added to the master mix now instead of to individual tubes during Step 9. Increase the aliquot in Step 7 as necessary.
6. Label a 12-tube strip and cut it in half so it will fit in a picofuge.
7. Add 17 μL (core TagSet only) or 24 μL (core TagSet plus Extension TagSet) of master mix to each of the 12 tubes using a fresh pipette
tip for each well.
8. Add RNA according to the sample type as follows:
a. If using total RNA: Add total RNA sample (maximum volume of 13 μL for core TagSet only, or 6 μL for core TagSet plus Extension TagSet) to each tube. NanoString recommends starting with 50-100 ng of total RNA per hybridization reaction and performing a titration to determine the optimal lower limit.
b. If using cell lysates: Add cell lysate sample (maximum volume of 3 μL) to each tube. NanoString recommends starting with 5,000-10,000 cells per hybridization reaction and performing a titration to determine the optimal lower limit.
IMPORTANT: If using cell lysates, adding more than 3 μL of sample will have an adverse impact on hybridization efficiency
of some probes due to the denaturing effect of the lysis buffer. The same amount of lysate should be used when comparing
samples.
9. If necessary, add nuclease-free water to each tube to bring the volume of each reaction to 30 µL.
10. Program the thermal cycler to use a 30 μL volume, calculated temperature, and heated lid. Set at 67°C for the selected hybridization
time (typically 16-21 hours; see Step 11) and then ramp down to 4°C. To minimize the potential for evaporation, the thermal cycler lid
should be set at 5° above the block temperature. Cap tubes and mix the reagents by inverting the strip tubes several times and flicking
with a finger to ensure complete mixing. Briefly spin down the hybridization reactions at less than 1,000 rpm and immediately place
the strip tubes in the thermal cycler.
11. Incubate reactions for at least 16 hours. Maximum hybridization time should not exceed 48 hours. Ramp reactions down to 4°C and
process the following day. Do not leave the reactions at 4°C for more than 24 hours or increased background may result.
NOTE: The purpose of selecting a fixed hybridization time followed by a ramp down to 4°C is to ensure equivalent hybridization times of all
assays being directly compared in the same series of experiments. Hybridization efficiency improves with time, and target counts may
increase 5% per hour between 16 and 24 hours. Although a 16-hour incubation is adequate for most purposes, a longer incubation will
typically increase counts.
Molecules That Count®
Gene Expression
–
Copy Number Variation
–
Single Cell Gene Expression
11
nCounter Elements™ General Purpose Reagents
3
DNA Hybridization Guidelines
Overview
nCounter Elements technology can be used to detect DNA for purposes such as determining copy number variation (CNV) and performing
counts of genetic loci in enriched DNA. Reagents are compatible with a wide variety of DNA sample types. However, additional sample
processing steps are required before assaying most types of DNA samples. Read these important considerations before performing the
hybridization protocol if using nCounter Elements reagents with DNA samples. If using samples that are not addressed in this manual, contact
customer support for additional information ([email protected]).
Fragmentation
Purified genomic DNA is typically larger in size (> 20 kb) than the average mRNA (~1.5 kb). To ensure adequate hybridization kinetics, DNA
must be fragmented prior to hybridization and analysis. In the case of immunoprecipitated material (ChIP-string assays), the DNA has
typically been fragmented during the IP process and additional fragmentation is not required.
Two methods of fragmentation are recommended: digestion by the Alu1 restriction enzyme, or Covaris AFA™-based fragmentation. When
using high molecular weight genomic DNA from cell lines, blood, or fresh or frozen tissue, NanoString recommends using Alu1-based
fragmentation. Digestion of human genomic DNA with Alu1 results in an average fragment size of 500 bases. Optimal target fragments for
hybridization (~100-750 bases) are selected during the probe design process.
When using degraded genomic DNA (e.g., from FFPE samples) NanoString recommends using Covaris AFA™-based fragmentation, although
Alu1 can also be used. The optimal target size is 200-300 bases. It may also be possible to use standard sonication or chemical or mechanical
fragmentation methods if fragment length is controlled adequately. For best results, all samples (including the reference sample) should have
similar fragmentation profiles.
CNV Reference Sample Considerations
Genomic DNA from a reference sample may be used to determine copy number with nCounter Elements. Reference samples should not
contain copy number variations in the test regions. For best results, select one or more genomic DNA reference samples that are of the same
sample type as the test samples (e.g., FFPE, cell line, or blood) and have been purified and fragmented in the same way as the test samples.
The reference DNA(s) should be run at least once whenever the content of the CodeSet is changed (i.e., different loci are being measured).
Please review detailed guidelines and suggested protocols for fragmenting DNA samples in the nCounter DNA Assay User Manual.
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Denaturation
DNA is typically double-stranded in its biological conformation, whereas RNA is single-stranded. Double-stranded DNA must be denatured
prior to hybridization.
IMPORTANT: NanoString recommends a denaturation temperature of 95°C for 5 minutes, followed by an immediate ramp down
to 4°C or quick cooling on ice. Incomplete denaturation results in decreased sensitivity.
DNA Input Amount
The recommended mass of input genomic DNA for most hybridization reactions is 150-300 ng, three times greater than the mass of input
RNA recommended as a starting point when measuring gene expression. This is due to the fact that most non-repetitive genomic DNA
sequences are present at two copies per cell in normal diploid samples. In contrast, the amount of a given RNA can range from one to many
thousands of copies per cell. The maximum sample input volume for DNA hybridization is 13 μL using a core TagSet, and 6 μL if an Extension
TagSet is added. Thus, the sample DNA concentration must be high enough to provide an adequate mass.
If the available sample is not a limiting factor, increasing the DNA input amount to 600 ng may provide better resolution for small variations
in CNV assays and increase accuracy for highly degraded FFPE samples.
For DNA that has been enriched (e.g., via immunoprecipitation, exome capture, or target enrichment) the input requirements are typically
lower than those required for standard genomic DNA. For these sample types, recommended starting input amounts are 5-50 ng depending
on the level of enrichment. Input amounts may need to be optimized for some experimental conditions.
DNA Quality
DNA must be free of contaminating RNA for accurate copy number analysis. NanoString strongly recommends that DNA preparations be
RNAse-treated as described by the extraction kit manufacturer. RNA contamination may negatively impact the quality of the data in two
ways:
1. Even when probes are selected to non-coding sequences, the presence of unprocessed RNA may result in artificially increased signal
and copy number determinations.
2. RNA may result in over-estimation of DNA concentration when measured by UV absorbance, leading to lower than recommended
DNA input amounts and lower counts. For pure genomic DNA preparations, NanoString recommends A260/280 ratios between 1.7
and 1.9 and A260/230 ratios between 1.3 and 2.0. Fluorescence-based assays using dyes specific for DNA may provide the most
accurate concentration measurements if RNA contamination is suspected.
DNA extracted from FFPE samples is typically degraded due to the fixation and storage process. NanoString recommends that the average
size of extracted DNA be greater than 1 kb prior to fragmentation.
Molecules That Count®
Gene Expression
–
Copy Number Variation
–
Single Cell Gene Expression
13
nCounter Elements™ General Purpose Reagents
PACKAGE INSERT
IMPORTANT: Do not vortex or pipet vigorously as this may shear the Reporter Tags. Mix only by flicking or inverting tubes. A
picofuge is preferable when spinning down solutions due to its low speed. If using a microfuge, do not “pulse” the machine as
it may reach maximum speed and spin the TagSet out of solution. Spin at less than 1,000 rpm for no more than 30 seconds.
NOTE: The following guidelines cover general steps for setting up DNA hybridization reactions. Each specific application using nCounter
Elements GPRs will require optimization and validation using appropriate performance metrics defined by the end user.
All reagents are provided for multiples of 12 reactions. Scale up the suggested volumes for additional reactions. Keep in mind that these
instructions for 12 reactions use a multiplier of 13 to allow for one reaction’s worth of dead volume in the master mix.
The final hybridization reaction will have a volume of 30 μL and contain the following components in each tube: 10 μL of hybridization buffer,
5 μL of TagSet, 1 μL of 30X Working Probe A Pool (0.6 nM each Probe A), 1 μL of 30X Working Probe B Pool (3 nM each Probe B), and up to
13 μL of sample DNA (Figure 3.1). If using an Extension TagSet, also add 5 μL of the Extension TagSet, 1 μL of the 30X Extension Probe A Pool
and 1 μL of 30X Extension Probe B Pool, and reduce the total volume of sample to no more than 6 μL (Figure 3.1). Instructions on preparing
30X Working Probe Pools are provided in Chapter 4.
FIGURE 3.1: Guide to creating master mix and preparing DNA hybridization reactions.
Dilute Master Probe Stocks to 30X Working Probe Pools
30X Working Probe A Pool
30X Working Probe B Pool
TagSet
Hybridization Buffer
Aliquot master mix to each tube
Add sample DNA to each tube (see guidelines)
Add nuclease-free water, if needed, to reach a
final volume of 30 μL
Hybridization reactions at 67°C for at least 16 hours
Each Reaction
13 Reactions
Hybridization Buffer
10 μL
130 μL
TagSet
5 μL
65 μL
30X Working Probe A Pool
1 μL
13 μL
30X Working Probe B Pool
1 μL
13 μL
Sample*
Up to 13 μL
Total Volume
30 μL
Each Reaction
13 Reactions
Hybridization Buffer
10 μL
130 μL
TagSet
5 μL
65 μL
Extension TagSet
5 μL
65 μL
30X Working Probe A Pool
1 μL
13 μL
30X Extension Probe A Pool
1 μL
13 μL
30X Working Probe B Pool
1 μL
13 μL
30X Extension Probe B Pool
1 μL
13 μL
Sample*
Up to 6 μL
Total Volume
30 μL
*NanoString recommends preparing samples with a high concentration
to facilitate reducing the sample volume if an extension TagSet is later
necessary.
IMPORTANT: Master Probe Stocks must be diluted to 30X Working Probe Pools (see page 18). Addition of concentrated Master
Probe Stocks directly to the assay will result in assay failure.
14
NanoString® Technologies
PACKAGE INSERT
1. See the directions in Chapter 4 to create the 30X Working Probe Pools. The accuracy of the probe concentration in the hybridization
reaction is important for maximizing sensitivity. Be sure to dilute the pools to the appropriate 30X concentration before addition to
the master mix below.
2. Remove an aliquot of the TagSet from the freezer and thaw it on ice. Invert several times to mix well, and briefly spin down the reagent
at less than 1,000 rpm.
3. Each TagSet tube contains 65 μL of reagent. Create a master mix by adding reagents directly into the TagSet tube. First add 130 μL
of hybridization buffer, followed by 13 μL of the 30X Working Probe A Pool. Mix well by flicking the tube and briefly spin down at less
than 1,000 rpm before adding 13 μL of the 30X Working Probe B Pool. Mix and spin again.
a. If using an Extension TagSet, also add 65 μL of the Extension TagSet reagent and 13 μL of the 30X Extension Probe A Pool before mixing. Then also add 13 μL of the 30X Extension Probe B Pool before the final mixing step.
b. If all samples have the same volume, additional nuclease-free water may be added to the master mix now instead of to individual tubes during Step 8. Increase the aliquot in Step 5 as necessary.
4. Label a 12-tube strip and cut it in half so it will fit in a picofuge.
5. Add 17 μL (core TagSet only) or 24 μL (core TagSet plus Extension TagSet) of master mix to each of the 12 tubes using a fresh pipette
tip for each well.
6. Denature DNA samples at 95°C for 5 minutes, followed by an immediate ramp down to 4°C or quick cooling on ice.
7. Add fragmented and denatured DNA sample (maximum volume of 13 μL for core TagSet only, or 6 μL for core TagSet plus Extension
TagSet) to each tube. See the above sections titled “Fragmentation” and “DNA Input Amount” for guidelines. If the sample was
isolated by ChIP, fragmentation is not necessary
8. If necessary, add nuclease-free water to each tube to bring the volume of each reaction to 30 μL.
9. Program the thermal cycler to use a 30 μL volume, calculated temperature, and heated lid. Set at 67°C for the selected hybridization
time (typically 16-21 hours; see Step 10) and then ramp down to 4°C. To minimize the potential for evaporation, the thermal cycler lid
should be set at 5° above the block temperature. Cap tubes and mix the reagents by inverting the strip tubes several times and flicking
with a finger to ensure complete mixing. Briefly spin down the hybridization reactions at less than 1,000 rpm and immediately place
the strip tubes in the thermal cycler.
10. Incubate reactions for at least 16 hours. Maximum hybridization time should not exceed 48 hours. Ramp reactions down to 4°C and
process the following day. Do not leave the reactions at 4°C for more than 24 hours or increased background may result.
NOTE: The purpose of selecting a fixed hybridization time followed by a ramp down to 4°C is to ensure equivalent hybridization times of all
assays being directly compared in the same series of experiments. Hybridization efficiency improves with time, and target counts may
increase 5% per hour between 16 and 24 hours. Although a 16-hour incubation is adequate for most purposes, a longer incubation will
typically increase counts.
Molecules That Count®
Gene Expression
–
Copy Number Variation
–
Single Cell Gene Expression
15
nCounter Elements™ General Purpose Reagents
4
Creating Oligonucleotide Probe Pools
Overview
The oligonucleotide probes must be formatted into two separate pools, one containing all Probe A’s (Master Probe A Stock), and one
containing all Probe B’s (Master Probe B Stock). If stored in aliquots at -20°C to -80°C, the Master Probe Stocks can be used for many
experiments. Please refer to your oligonucleotide supplier for specific storage recommendations and shelf-life information.
Before setting up the hybridization reactions, 30X Working Probe Pools must be generated by diluting each Master Probe Stock. The final
concentration of the oligonucleotides in 30X Working Probe A Pool will be 0.6 nM each, and the final concentration of the oligonucleotides
in 30X Working Probe B Pool will be 3 nM each. The absolute concentration of the pool will vary depending on the plex of the experiment;
due to the dilute DNA concentrations of many 30X Working Probe Pools, long-term storage and reuse of these pools is not recommended.
The protocols below provide an example of how to generate the Master Probe Stocks and 30X Working Pools for Probe A and Probe B.
Depending on the oligonucleotide format obtained from the supplier, different pipetting volumes and dilutions may be necessary to achieve
the required final concentrations.
IMPORTANT: The concentrations of Probe A and Probe B in the hybridization reaction are critical for maximizing the sensitivity
of the assay. Be sure to follow appropriate pooling and dilution protocols carefully to create accurate 30X Working Probe Pools.
16
NanoString® Technologies
PACKAGE INSERT
Creating Master Probe Stocks
NOTE: Some oligo suppliers will provide pooled oligos which can be used in place of creating your own Master Probe Stocks. If utilizing this
service, specify a pool of Probe A’s at a final concentration of 5 nM per oligo, and a separate pool of Probe B’s at a final concentration
of 25 nM per oligo. Pooled probes provided by oligo supplies at the recommended concentrations must still be further diluted to
create 30X Working Probe Pools (see next page) before addition to the assay.
IMPORTANT: Always create separate Probe A and Probe B stocks. Do NOT create a combined Master Probe Stock containing
Probe A’s and Probe B’s in the same tube; elevated background and lowered assay sensitivity may result.
1. NanoString recommends resuspending individual Probe A oligonucleotides at a 1 μM concentration, and individual Probe B
oligonucleotides at a 5 μM concentration. Oligonucleotides should be resuspended in TE (10 mM Tris pH 8, 1 mM EDTA) or a similar
buffer and stored frozen under conditions recommended by supplier. To prepare Master Probe Stocks, begin by removing the
appropriate Probe A and Probe B oligonucleotides from the freezer and thawing on ice.
2. Master Probe A Stock
a. Pipet 5 μL of each Probe A (starting concentration 1 μM) into a 1.7 mL microfuge tube.
b. Add TE to a final combined volume of 1 mL.
c. The final concentration of each Probe A in the Master Probe A Stock will be 5nM.
d. Store in aliquots at -20°C or -80°C as recommended by supplier.
3. Master Probe B Stock
a. Pipet 5 μL of each Probe B (starting concentration 5 μM) into a 1.7 mL microfuge tube.
b. Add TE to a final combined volume of 1 mL.
c. The final concentration of each Probe B in the Master Probe B Stock will be 25nM.
d. Store in aliquots at -20°C or -80°C as recommended by supplier.
NOTE: The probes in the Master Probe Stocks must be appropriate for the targets being queried. If reporter tags are reassigned to new
targets, new Master Probe Stocks containing the specific set of appropriate probes must be created. Do NOT add additional probes
to existing Master Probe Stocks. If using an Extension Tagset as well as a Core TagSet, create separate Master Probe Stocks for the
Extension Probes.
IMPORTANT: Minimize freeze-thaw cycles by storing Master Probe Stocks in appropriate aliquots at -20°C or -80°C. Thaw each
aliquot only once and then place at 4°C for use in creating multiple 30X Working Probe Pools. A suitable aliquot size for your
workflow can be calculated from the information in Table 4.1 ( see page 18) based on your expected assay throughput. Follow
supplier’s guidance on stability of the oligonucleotide stocks at 4°C.
Molecules That Count®
Gene Expression
–
Copy Number Variation
–
Single Cell Gene Expression
17
nCounter Elements™ General Purpose Reagents
PACKAGE INSERT
Creating 30X Working Probe Pools
IMPORTANT: Always create separate Probe A and Probe B 30X Working Probe Pools. Do NOT create a combined 30X Working
Probe Pool containing Probe A’s and Probe B’s in the same tube; elevated background and lowered assay sensitivity may result.
1. 30X Working Probe A Pool
a. Determine the number of assays to be performed. Starting with the Master Probe A Stock, follow the 8.3-fold dilution outlined in Table 4.1 to generate a 30X Working Probe A Pool at the appropriate scale.
b. Mix well and spin to bring contents to the bottom of the tube. The final concentration of each Probe A in the 30X Working Probe A Pool will be 0.6 nM.
c. Proceed to the Hybridization Guidelines for RNA or DNA described in Chapters 2 and 3. The final concentration of each Probe A in the 30 μL hybridization assay will be 20 pM.
d. Due to the dilute DNA concentrations of many 30X Working Probe Pools, long-term storage and reuse of these pools is not recommended. A fresh dilution of the Master Probe Stock should be made for subsequent assays.
2. 30X Working Probe B Pool
a. Determine the number of assays to be performed. Starting with the Master Probe B Stock, follow the 8.3-fold dilution outlined in Table 2.1 to generate a 30X Working Probe B Pool at the appropriate scale.
b. Mix well and spin to bring contents to the bottom of the tube. The final concentration of each Probe B in the 30X Working Probe B Pool will be 3 nM.
c. Proceed to the Hybridization Guidelines for RNA or DNA described in Chapters 2 and 3. The final concentration of each Probe B in the 30 μl hybridization assay will be 100 pM.
d. Due to the dilute DNA concentrations of many 30X Working Probe Pools, long-term storage and reuse of these pools is not recommended. A fresh dilution of the Master Probe Stock should be made for subsequent assays.
TABLE 4.1: Guide to diluting Master Probe Stocks to generate 30X Working Probe Pools. Due to the dilute nature of the final pool, use of TE-Tween
(10 mM Tris pH 8, 1 mM EDTA, 0.1% Tween®-20) is recommended.
Number of Assays
12
24
36
48
60
72
84
96
108
120
132
144
156
168
180
192
18
Aliquot from Master Probe Stock (μL)
4
4
5
7
8
10
11
13
15
16
18
19
21
23
24
26
TE-Tween (μL)
29
29
37
51
59
73
81
95
110
117
132
139
154
169
176
191
Final Volume (μL)
33
33
42
58
67
83
92
108
125
133
150
158
175
192
200
217
PACKAGE INSERT
5
Evaluating Performance
Each individual laboratory should evaluate the performance of nCounter Elements to ensure that it is adequate for the specific application
for which the reagents are being used. Appropriate performance metrics should be defined based on the desired attributes of the final
assay. Below are examples of analyses that may be performed to evaluate reproducibility, compare responses to changing sample input
concentration, and correct for lot-to-lot variability. Note that these examples are not meant to imply specific performance characteristics of
nCounter Elements reagents; results may vary depending on assay design, sample input, or other factors.
Technical Replicates
In order to determine the reproducibility of an assay utilizing nCounter Elements reagents, two replicates assaying the same purified RNA
sample can be hybridized to an nCounter Elements TagSet and associated target-specific probes (Probe A and Probe B). Replicates can be
plotted in linear space with the first replicate on the y-axis and the second replicate on the x-axis. Linear regression can then be performed to
generate an R2 value indicating the degree of correlation between the two replicates. In the example shown in Figure 5.1, technical replicates
on 100 ng of target RNA were run in a 192-Plex hybridization and compared as described above.
Replicate 1 Counts
100000
R2 = 0.99
10000
1000
100
10
1
1
10
100
1000
10000
100000
Replicate 2 Counts
FIGURE 5.1: Correlation between two hybridization assays using the same RNA sample.
Molecules That Count®
Gene Expression
–
Copy Number Variation
–
Single Cell Gene Expression
19
nCounter Elements™ General Purpose Reagents
PACKAGE INSERT
Sample Input Titration
Counts – 200, 300, and 500 ng
Input Total RNA
In order to evaluate the response of assays utilizing nCounter Elements reagents to varying amounts of sample input, comparisons can
be made by titrating sample inputs. Data from one sample can be defined as the baseline value to which the others are compared. Linear
regression can then be applied to determine a slope value that should correspond to the concentration of the test sample relative to the
baseline sample. In the example shown in Figure 5.2, normalized counts from total RNA input amounts of 100, 200, 300, and 500 ng of
the same sample are plotted. The 100 ng assay is used as the baseline value on the x-axis against which the others are plotted. The slopes
correlate closely with the expected values of 2, 3, and 5 with 99% correlation, indicating a linear response to increasing target concentration.
Counts – 100 ng Input Total RNA
FIGURE 5.2: Linear regression analyses of a sample titration.
20
NanoString® Technologies
PACKAGE INSERT
Qualifying Probes to Confirm Low Background
Probe A and Probe B oligonucleotides can be evaluated for their contribution to background in an Elements assay. To do this, the Probe
A and B oligonucleotides can be assessed separately and together using no-sample control hybridizations under standard conditions. An
example of how to characterize probe-associated background levels is outlined below.
Low background levels can be verified and, in the case where higher than expected background is observed, the causative reagent can be
identified by performing hybridizations with the components shown in Table 5.1.
TABLE 5.1: Example of an experimental design for evaluating background
Assay Purpose
Probe A
Asses background contributions intrinsic to TagSet
Asses background contributions of Probe A and Probe B
Positive Control
Probe B
Sample
–
–
–
+
–
–
–
+
–
+
+
–
+
+
+
NanoString demonstrated this strategy by running a 36-plex Elements CodeSet in duplicate in the five assays described above. Hybridizations
were performed at 67°C for 16-20 hours and purified using an nCounter Prep Station. Cartridges were scanned using an nCounter Digital
Analyzer. Raw counts were normalized to the ERCC positive controls and average normalized counts for the duplicate reactions for each
target are shown below.
Comparing background levels observed with a larger set of historical data (Table 5.2) to the background observed for the 36-Plex CodeSet
(Table 5.3) shows that background for this 36-plex CodeSet falls within the range established by historical data. The positive control, in this
case Universal Human Reference RNA (UHR), shows that the level of expression for these genes when using 100 ng of input RNA ranges from
60 to over 80,000 counts, well above the background signal.
NOTE: The exact level of background will depend on the instrument and protocol used for scanning (e.g., FOV number), the number of
reporter tags in the CodeSet and, in rare cases, the sequence of individual Probe A and B molecules.
TABLE 5.2: Historical data for background contributed by various Probe A and Probe B designs
Probe A Only
Probe B Only
Probe A + Probe B
Average Counts
1
2
3
Standard Deviation
1
2
3
Maximum Counts
18
14
27
Number of Probes Tested
401
401
593
Molecules That Count®
Gene Expression
–
Copy Number Variation
–
Single Cell Gene Expression
21
nCounter Elements™ General Purpose Reagents
PACKAGE INSERT
TABLE 5.3: Counts observed for 36-Plex Elements CodeSet assays following the experimental design outlined in Table 5.1
22
No Sample
100 ng UHR
Tag ID
Gene
tag-001
Gene 1
1
4
1
2
80,561
tag-002
Gene 2
0
2
1
1
586
tag-003
Gene 3
0
7
1
5
1,716
tag-004
Gene 4
0
1
0
1
6,217
tag-005
Gene 5
1
3
1
4
797
tag-006
Gene 6
1
2
1
3
728
tag-007
Gene 7
1
6
2
4
364
tag-008
Gene 8
2
2
1
2
131
tag-009
Gene 9
1
2
3
2
879
tag-010
Gene 10
1
4
2
6
684
tag-011
Gene 11
0
2
1
2
210
tag-012
Gene 12
1
8
1
12
100
tag-013
Gene 13
0
1
1
2
248
tag-014
Gene 14
0
1
2
2
158
tag-015
Gene 15
2
1
3
1
1,398
tag-016
Gene 16
2
3
1
3
501
tag-017
Gene 17
1
1
2
1
666
tag-018
Gene 18
1
2
2
3
111
tag-019
Gene 19
1
2
1
3
1,070
tag-020
Gene 20
1
0
0
0
1,014
tag-021
Gene 21
1
2
3
3
1,887
tag-022
Gene 22
0
2
1
4
91
tag-023
Gene 23
1
3
2
3
373
tag-024
Gene 24
0
1
1
0
490
tag-025
Gene 25
2
2
0
3
214
tag-026
Gene 26
1
4
0
1
658
tag-027
Gene 27
2
0
3
0
187
tag-028
Gene 28
1
1
1
1
60
tag-029
Gene 29
2
4
4
3
415
tag-030
Gene 30
1
2
3
4
590
tag-031
Gene 31
1
2
2
1
367
tag-032
Gene 32
0
1
0
2
170
tag-033
Gene 33
0
1
2
2
143
tag-034
Gene 34
0
2
1
2
177
tag-035
Gene 35
0
1
4
3
108
tag-036
Gene 36
2
2
2
4
315
No Probe A or Probe B
Probe A Only
Probe B Only
Probe A + Probe B
Probe A + Probe B
NanoString® Technologies
PACKAGE INSERT
Calculating Correction Factors Between Lots
In order to evaluate variation between lots of nCounter Elements GPRs and calculate correction factors, comparisons can be made between
one or more lots of Elements reagents run on the same reference sample. Results from new lots can be plotted against older lots and linear
regression applied to determine R2 values quantifying the correlation between lots. For each individual gene or target being analyzed, a
correction factor to enable normalization between lots can be calculated by dividing the value (number of counts) obtained for each target
using Elements Lot 1 (reference lot) with the value obtained using Lot 2. When Lot 2 is run on an experimental sample, each datapoint can
be multiplied by its correction factor to create a dataset normalized to Lot 1.
In the Figure 5.3, three lots (Lots 1-3) of 36-plex nCounter Elements reagents were compared (FIgure 5.3A) and correction factors were
created using Lot 1 as the reference lot (Figure 5.3B) and applied to experimental data generated with Lots 2 and 3 (Figure 5.3C) as described
above.
FIGURE 5.3: Uncorrected plots comparing Lot 1 with Lots 2 and 3 (A); correction factors (B); and corrected plots comparing Lot 1 with Lots 2 and 3 (C).
Molecules That Count®
Gene Expression
–
Copy Number Variation
–
Single Cell Gene Expression
23
nCounter Elements™ General Purpose Reagents
PACKAGE INSERT
Manufacturer
Consult Instructions for Use
Catalogue or Reference Number
Batch Code / Lot Number
Temperature Range Storage Conditions
Lower Limit of Temperature Storage Conditions
Upper Limit of Temperature Storage Conditions
For Use By / Expiry Date
Date of Manufacture
Room Temp. = Room Temperature
HYB = Hybridization
Regulatory Disclaimer
FOR LABORATORY USE.
© 2013 NanoString Technologies, Inc. All rights reserved. NanoString®, NanoString Technologies®, nCounter®, Molecules That Count®,
nDesign™, and nCounter Elements™ are registered trademarks or trademarks of NanoString Technologies, Inc., (“NanoString”) in the
United States and/or other countries.
Changes to Previous Versions
24
Version Number
Description
LBL-C0266-01
Initial release
LBL-C0266-02
Adjusted probe-pooling guidance
LBL-C0266-03
Adjusted guidance for performing
hybridizations with RNA lysates and added
section on verifying olignoucleotide probes
PACKAGE INSERT
nCounter Elements™ General Purpose Reagents
NanoString Technologies, Inc.
CONTACT US
530 Fairview Ave N
Suite 2000
Seattle, Washington 98109
[email protected]
United States:[email protected]
Tel: (888) 358-6266
Europe:[email protected]
Fax: (206) 378-6288
Other Regions:[email protected]
SALES CONTACTS
www.nanostring.com
© 2013 NanoString Technologies, Inc. All rights reserved. NanoString®, NanoString Technologies®, nCounter®, Molecules That Count®, nDesign™, and nCounter Elements™ 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
that appear in this document are the property of their respective owners. The manufacture, use and/or sale of NanoString product(s) may be subject to one or more patents or pending patent
applications owned by NanoString or licensed to NanoString from Life Technologies Corporation and other third parties.
FOR LABORATORY USE.
LBL-C0266-03