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System Design
System Performance
- What is the dynamic range of the system?
- How is the nCounter Analysis System quantitative?
- What is the threshold of detection (or sensitivity) for the nCounter Analysis System?
- Can nCounter measure fractional fold changes and over what range?
- What is the average %CV at one copy per cell expression level?
- What is the maximum number of genes that can be analyzed at once?
Applications
Sample Types and Starting Material
System Design
What is unique about the nCounter Analysis System?
The nCounter System offers unparalleled performance in digital gene expression by offering a combination of unique features: multiplexing, high sensitivity and ease-of-use. Users can currently analyze the expression levels of up to 550 genes simultaneously, with sensitivity comparable to quantitative PCR systems and with less than 15 minutes of hands on time per reaction.
Return To TopWhat is the technology at the heart of the nCounter System?
The nCounter System uses direct digital detection for gene expression analysis. Color coded molecular barcodes, called Reporters, and single molecule imaging are used to detect and count hundreds of unique transcripts in a single reaction.
Return To TopWhat is digital detection?
In contrast to analog detection, digital detection does not rely on relative levels of signal, such as fluorescence, for detection and quantification. Instead, digital detection relies on discrete units for measurement. In the case of NanoString technology, digital detection is used on two levels. First, each transcript is detected by a probe bound to a segment of DNA that is attached to a unique string of colored fluorophores (the molecular barcode). Identification of that transcript therefore depends only on the order of fluors on the string, rather than intensities of the fluors. Secondly, the number of total transcripts in a sample is quantified by counting the total number of times a particular string of fluors (barcode) is detected.
Return To TopWhat is direct detection?
Direct detection refers to the detection of a molecule of interest directly without conversion to a different type of molecule or amplification of that molecule. Unlike microarrays or PCR based gene expression analysis technologies, our system does not rely on synthesis of a cDNA strand or PCR amplification. Therefore, no enzymes are used in our procedure. Instead, our barcode-labeled probes anneal directly to mRNAs in solution, and the hybrid molecule is then immobilized, detected and counted.
Return To TopWhat is the system throughput?
The system can process up to 72 samples in 24 hours. At the 550-plex scale, this corresponds to 39,600 data points per day.
Return To TopWhat is the time to result?
The entire nCounter assay takes 22 hours for one 12-sample cartridge. This includes 16 hours (overnight hybridization can be up to 30 hours to accommodate the researcher’s schedule) for the hybridization, two hours on the liquid-handling Prep Station, and four hours on the Digital Analyzer. Since the Prep Station and Digital Analyzer units are completely automated, the total hands-on time for the user is less than 15 minutes. Note that this is independent of the number of genes analyzed: the assay time required for 20 genes is the same as for 550 genes.
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System Performance
What is the dynamic range of the system?
We have shown that the system can accurately measure up to six logs of reporter counts during experiments designed specifically to measure dynamic range. However, with rare exception, this dynamic range is not necessary to measure biological levels of expression. The nCounter System requires no amplification of the target mRNA; therefore, the dynamic range necessary to measure the full range of expression in cells is simply the biological range of expression, typically three orders of magnitude.
Return To TopHow is the nCounter Analysis System quantitative?
The nCounter Analysis System is able to detect, count and report molecules across a wide range of expression levels. The nCounter CodeSets include positive control codes and synthetic RNA targets that have been spiked in to the CodeSet at concentrations ranging from 0.1fM -100fM which roughly correspond to 0.2-200 copies per cell of mRNA. Using these internal controls, users can plot a regression line and quantify relative levels of expression for each of the genes in the CodeSet. The nCounter specifications are based on an input of 100ng of total RNA.
Return To TopWhat is the threshold of detection (or sensitivity) for the nCounter Analysis System?
NanoString guarantees detection of 10,000 molecules (which equals approximately one copy per cell) over 90% of the time when using our standard protocol; however we routinely detect as few as 2000 molecules in a sample (which equals approximately 1 copy per 5 cells).
Return To TopCan nCounter measure fractional fold changes and over what range?
Yes, we can measure as little as 1.2-fold changes of a single transcript at 20 copies per cell (10fM) with statistical significance (p<0.05). For genes expressed at levels between 0.5 and 20 copies per cell, we routinely detect 1.5-fold differences in expression levels with the same level of confidence.
Return To TopWhat is the average %CV at one copy per cell expression level?
We routinely see %CVs (Coefficient of Variation) in the one copy per cell expression range at ~10% and even lower for more highly expressed genes.
Return To TopWhat is the maximum number of genes that can be analyzed at once?
We currently offer CodeSets that multiplex 550 endogenous transcripts in a single assay. We expect this number to increase in the near future.
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Applications
What kind of projects are ideal for the nCounter Analysis System?
We expect the nCounter system to be extremely useful for researchers interested in validating microarray expression results, biomarker analysis, gene expression experiments involving hundreds of genes, and users wanting gene expression results from large numbers of samples.
Return To TopCan you measure transcript variants?
Yes, in many cases we can detect transcript variants.
Return To TopCan I profile microRNAs on the nCounter Analysis System?
Currently, the system does not support miRNA profiling. This is a project that is in development at NanoString Technologies.
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Sample Types and Starting Material
What sample types can be used on the nCounter Analysis System?
The nCounter gene expression assay can use purified total RNA, raw cell lysates in guanidinium salts, blood lysate from PAXGene™ and amplified RNA directly input into the hybridization reaction. We have also obtained good results from total RNA isolated from FFPE samples, provided the RNA meets minimal integrity requirements (more than 50% greater than 300bp). Finally, we have preliminary evidence that suggests that total RNA purified from PAXGene tubes can be used in our system.
Return To TopHow much starting material do you need?
We suggest 100ng of total RNA, or a lysate of 10,000 cells. More or less material may be used to boost signal or reduce sample requirements, but quantitation of extremely rare transcripts may be affected when less material is used in our system.
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Data Analysis
How do you normalize the data?
We recommend normalizing data from the nCounter CodeSet first using the internal system controls to remove any slight assay-to-assay system variation and then to 3-4 validated reference genes in your endogenous samples.
Return To TopCan I compare the nCounter System with my microarray data?
Yes. The fold change data obtained from an nCounter analysis correlates well with fold change results obtained from microarray analyses. The level of concordance between the nCounter and microarray results is similar to comparisons of different microarray platforms.
Return To TopCan nCounter data be used together with qPCR data?
Yes, we’ve found that there is an excellent correlation between nCounter analyses and qPCR analyses, both in terms of relative expression levels and fold changes. Moreover, the multiplexing capabilities of nCounter analyses increase the efficiency with which data can be obtained at qPCR levels of sensitivity. We therefore recommend using nCounter analyses to extend your current set of qPCR data.
Return To TopHow do I analyze my data?
The comma separated value file returned by the nCounter system is easily imported and analyzed in other programs, including Excel, Open Office, or Matlab. We provide suggested guidelines for normalization and analysis of data from the nCounter Analysis System. See: nCounter Data Analysis Guidelines for more information.
Return To TopIs there documentation available about analyzing my data?
Yes, there is a data analysis guidelines document that is available in the Product Literature section of this website.
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