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Below you will find various nCounter® information, content, and graphics to assist you in writing grants or preparing presentations. NanoString authorizes free access to this content for use in grants and public presentations.

INSTRUCTIONS: To use the text sections, simply highlight the text from the page, copy, and then paste into your source grant document. To use the images/graphs, right-click on the image, copy, and then paste into your grant document.

Overview of nCounter Analysis System

nCounter® Analysis System enables the profiling of hundreds of mRNAs, microRNAs, SNVs, CNVs, or Protein on one platform with high sensitivity and precision. The primary benefits of the platform are the ability to complete studies faster, with very high precision and using less precious sample material. Faster time to completion of studies is enabled by nCounter’s streamlined workflow, high sample throughput, and multiplexing capability. nCounter’s digital counting capability provides highly reproducible data over 5 logs of dynamic range and does not require any amplification steps that might introduce bias to the results. An additional advantage of nCounter chemistry is that it is highly tolerant of difficult sample types such as FFPE and crude-cell lysates. The utility of the nCounter system is demonstrated in over 1600 peer-reviewed publications, many in top journals. Click here to download the complete nCounter-featured publications list.

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. Specifically, molecular “barcodes” and single molecule imaging are employed to detect and count hundreds of unique targets in a single reaction. All nCounter instruments and reagents are provided by NanoString Technologies and are built under GMP/ISO13485 standards.

Introduction

The nCounter® Analysis System enables the profiling of hundreds of mRNAs, microRNAs, SNVs, CNVs or Protein on one platform with high sensitivity and precision. The primary benefits of the platform are the ability to complete studies faster, with very high precision and using less precious sample material. Faster time to completion of studies is enabled by nCounter’s streamlined workflow, high sample throughput, and multiplexing capability. nCounter’s digital counting capability provides highly reproducible data over 5 logs of dynamic range and does not require any amplification steps that might introduce bias to the results. An additional advantage of nCounter chemistry is that it is highly tolerant of difficult sample types such as FFPE and crude-cell lysates.

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. Specifically, molecular “barcodes” and single molecule imaging are employed to detect and count hundreds of unique targets in a single reaction. All nCounter instruments and reagents are provided by NanoString Technologies and are built under GMP/ISO13485 standards.


Technology Overview

NanoString’s nCounter technology is based on direct detection of target molecules using color-coded molecular barcodes, providing a digital count of the number of target molecules. The probe pair consists of a Reporter Probe, which carries the signal on its 5’ end, and a Capture Probe which carries a biotin on the 3’ end. The color codes carry six positions and each position can be one of four colors, thus allowing for a large diversity of codes that can be mixed together in a single reaction tube for direct hybridization to target and yet still be individually resolved and identified during data collection.

Capture and Reporter Probes (left) and, Probe pair bound to an ssDNA molecule (right)

Purification and binding of the hybridized complexes is carried out automatically on the nCounter Prep Station. Magnetic beads derivatized with short nucleic acid sequences that are complementary to the Capture Probe and the Reporter Probes are used sequentially. First, the hybridization mixture is allowed to bind to the magnetic beads by the Capture Probe. Wash steps are performed to remove excess Reporter Probes as well as DNA fragments that are not hybridized. After washing, the Capture Probes and Target/Probe complexes are eluted off of 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, the purified Target-Probe complexes are eluted off and are immobilized in the cartridge for data collection.

Data collection is carried out in the nCounter Digital Analyzer. Digital images are processed and the barcode counts are tabulated in a comma separated value (CSV) format.


Analysis of miRNA

miRNAs (at ~22 bp) require preparation to enable hybridization with nCounter probes which typically span 100 bp of target sequence. To prepare a sample containing miRNA molecules for hybridization in the nCounter assay, proprietary DNA sequences called miRtags are ligated to the mature miRNAs through complementarity with sequence-specific bridging oligonucleotides (bridges). This multiplexed reaction leads to the highly precise, sequence-specific tagging of all miRNAs in a single tube. Excess tags and bridges are removed via a simple enzymatic step in the same tube. No further purification is required. The miRtagged mature miRNA is then hybridized to a probe pair in the standard nCounter gene expression assay workflow. The Reporter Probe carries the signal; the Capture Probe allows the complex to be immobilized for data collection. Hybridization occurs in one multiplexed reaction.



Analysis of Genomic DNA

Before analyzing genomic DNA with nCounter, the DNA must be fragmented and denatured. The nCounter DNA Prep kit is supplied with all nCounter CNV assays to facilitate fragmentation. This kit contains the reagents necessary for fragmentation of genomic DNA via the restriction endonuclease, Alu I. Digestion of human genomic DNA with Alu1 results in an average of 500bp fragments and optimal target fragments for hybridization (~100–750 bases) are selected in probe design process. Other fragmentation methods are also compatible with the nCounter DNA assays. For the most accurate results in FFPE samples, we recommend fragmentation via Covaris AFA™ technology. The optimal target size range is 200-300 bases. It may also be possible to use chemical, standard sonication or mechanical fragmentation methods if fragment length is controlled adequately. For best results, all samples (including reference) should have similar fragmentation profiles.


nCounter® Prep Station


The nCounter Prep Station is the automated liquid handling component of the nCounter Analysis System. It processes samples post-hybridization to prepare them for data collection on the nCounter 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 cartridge for data collection.


nCounter® Digital Analyzer

The nCounter Digital Analyzer collects data by taking images of the immobilized fluorescent reporters in the sample cartridge with a CCD camera through a microscope objective lens. Images are processed internally and the data output files include the target identifier and count number along with a comprehensive tally of internal controls that allows each assay to be quantitative. The small data files can be distributed using a variety of methods and are easily integrated with commonly used data analysis and visualization packages.


nCounter® SPRINT

The nCounter® SPRINT is an all-in-one benchtop analyzer that combines liquid handling and imaging all-in-one. Hybridized samples are loaded into a SPRINT cartridge, where a series of microfluidic chambers purifies and then images the immobilized fluorescent reporters. Images are processed internally and the output files include gene or protein target name along with the associated count number.


nCounter® CodeSets

nCounter CodeSets are pools of color-coded molecular barcodes and capture probes designed for specific projects. In addition to target specific probes, a comprehensive mix of control probes are added to each CodeSet to quantitate, QC, and normalize data. Custom CodeSets and fixed-content panels are available for analysis of mRNA, miRNA, lncRNA, and DNA.


nCounter® Master Kits

The nCounter Master Kit contains all reagents, sample cartridges and consumables necessary for processing nCounter CodeSet Master Kits are packaged for processing 12, 48, or 192 assays.

Gene Expression

Sensitivity, Reproducibility, and Dynamic Range


Gene counts from two technical replicates plotted against one another demonstrate the assay reproducibility over a 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 is equal to a concentration of approximately 1 copy per cell. This data illustrates the high level of sensitivity and precision of the assay even at very low levels of expression.


Analysis of RNA Extracted from FFPE

FFPE-derived purified total RNA was input directly into the hybridization reaction and compared to a matched purified total RNA from fresh tissue as plotted on the x-axis. Results demonstrate that high quality data can be achieved from FFPE samples.


Direct Analysis of “Crude” De-paraffinized FFPE

This figure demonstrates that RNA extraction is often not necessary for analysis of FFPE samples. It shows the correlation of counts from “crude” FFPE extracts to counts from purified RNA from FFPE slices. FFPE extracts were prepared from a brain FFPE sample by removing paraffin with xylene and digesting proteins with Proteinase K.


Direct Analysis of Whole Blood Lysates

PaxGene lysed whole blood replicates were input directly into the hybridization reaction and compared to a matched purified total RNA sample as plotted on the x-axis. Results demonstrate that high quality data can be obtained by using PaxGene lysed whole blood.


Direct Analysis of Crude Cell Lysates

Raw cell lysate was input directly into the nCounter Analysis System hybridization reaction using approximately 2,500, 5,000 and 10,000 cells in lysis buffer and compared to 100ng of purified total RNA as plotted on the x-axis. Results were highly correlated with one another and demonstrate that comparable data can be achieved with either protocol.


miRNA

Reproducibility

To demonstrate the reproducibility of the miRNA assay, we used the nCounter Human miRNA Expression Assay Kit to analyze commercially purchased human total RNA. The samples were prepared and run on the system separately. When plotted against one another, the assay demonstrated very high reproducibility, with R2 values of greater than 0.99.


Analysis of RNA Purified from FFPE

Formalin-Fixed Paraffin-Embedded (FFPE) samples are difficult to analyze with many techniques due to RNA degradation. To demonstrate the ability of the miRNA assay to analyze degraded RNA samples, we tested FFPE-derived purified total RNA from liver samples against 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 (R2 > 0.95) between the two sample types.


Single-base Resolution

The Let7 family of miRNAs contains several members differing in sequence by as little as a single nucleotide. Percent cross-hybridization for the Let7 probes was assessed by tagging and hybridizing individual Let7 family miRNAs in the presence of the comprehensive human miRNA CodeSet including probes to eight Let7 family members. The minimal cross-hydridization observed (with no probe exceeding 6%) indicates that the nCounter miRNA assay can accurately discriminate similar sequence miRNAs with great specificity.


CNV

This figure demonstrates that nCounter generates linear response to increasing copy number. A synthetic DNA oligo was titrated at concentrations that are representative of varying copy numbers. Normalized counts are shown on the Y-axis, copy number is shown on the X-axis. The number of molecules counted increases linearly with an increase in copy number.


High Concordance With Hap-Map Data

This figure demonstrates that nCounter generates data that is highly concordant with publically available microarray data. A comparison of copy number calls between NanoString and microarray data for a single genomic region in 50 HapMap samples (Coriell Cell Repositories). Integer copy number values are shown relative to reference sample NA10851. The nCounter CNV assay was 100% accurate for 0, 1 and 2 copies in this region across all samples. Overall accuracy for the entire study (100 samples – 20 regions) was > 94%.


Analysis of DNA Purified from FFPE Samples

This figure shows copy number counts for genes included in the nCounter Cancer CN Assay CodeSet for 2 matched pairs of Fresh Frozen (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.