Frequently Asked Questions
Frequently asked questions are listed below (click the question to expand the answer).
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- What is unique about the nCounter Analysis System?
The nCounter Analysis System offers unparalleled performance in digital gene expression through a combination of unique features: multiplexing, high sensitivity, and ease-of-use. Users can analyze the expression of up to 800 genes simultaneously, with sensitivity comparable to quantitative PCR systems and requiring less than 15 minutes of hands-on time per reaction.
- What is the technology at the heart of the nCounter Analysis System?
The nCounter Analysis System uses direct digital detection for gene expression analysis. Color-coded molecular barcodes, called Reporter Probes, and single-molecule imaging are used to detect and count hundreds of unique transcripts in a single reaction.
- What 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's nCounter 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. Second, the number of total transcripts in a sample is quantified by counting the number of times a particular barcode is detected.
- What 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. 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.
- What is the system throughput?
The system can process up to 72 samples in 24 hours. At the 800-plex scale, this corresponds to 39,600 data points per day.
- What 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 800 genes.
- 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.
- How 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 - 100 fM 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.
- What 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).
- Can 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 (10 fM) 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.
- What is the average %CV at the expression level of one copy per cell?
We routinely see %CVs (coefficients of variation) in the one copy per cell expression range at ~10% and even lower for more highly expressed genes.
- What is the maximum number of genes that can be analyzed at once?
We offer CodeSets that multiplex 800 endogenous transcripts in a single assay.
- 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.
- Can you measure transcript variants?
Yes, in many cases we can detect transcript variants.
- Can I profile microRNAs on the nCounter Analysis System?
NanoString's nCounter technology is based on a novel method of direct molecular barcoding and digital detection of target molecules through the use of color-coded probe pairs. The nCounter miRNA Sample Preparation Kit provides reagents for ligating unique oligonucleotide tags (miRtags) onto the 3’ end of target miRNAs, allowing short RNA targets to be detected by nCounter probes. Currently we offer nCounter miRNA Expression Assay Kits for Human v3, Mouse v1.5, and Rat v1.5. We are in the process of phasing out Human v2 and earlier versions of Mouse and Rat. Additionally, we offer the nCounter à la carte miRNA CodeSets for analysis of subsets of miRNAs (20-50 targets) included in the latest species-specific miRNA Expression Assay Kits. Please contact an Account Manager to get a quote for your project, or email email@example.com.
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 added to 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 300 bp).
- How much starting material do you need?
We suggest 100 ng 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.
- Can I profile microRNAs on the nCounter Analysis System?
Currently we offer nCounter miRNA Expression Assay Kits for Human v3, Mouse v1.5, and Rat v1.5. We are in the process of phasing out Human v2 and earlier versions of Mouse and Rat. This product provides a sensitive, reproducible, and highly multiplexed method for detecting specific miRNAs within total purified RNA isolated from any source, including fresh or frozen cells and tissues, formalin-fixed paraffin embedded (FFPE) samples,and biological fluids (refer to the Technical Note, nCounter miRNA Analysis in Plasma and Serum Samples).
- Can 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.
- Can 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.
- How do I analyze my data?
The nSolver Analysis Software is a data analysis program that offers nCounter users the ability to quickly and easily QC, normalize, and analyze their data without having to purchase additional software packages. The nSolver software also provides seamless integration and compatibility with other software packages designed for more complex analyses and visualizations. It is free for all nCounter customers and available for download. Please sign in to our Customer Resources area to download the nSolver Analysis Software.
- Is there documentation available about analyzing my data?