NanoString's nCounter™ Analysis System utilizes a digital technology based on direct multiplexed measurement of nucleic acids using fluorescent barcodes that bind to these targets.
Assays for mRNA, miRNA and DNA are all possible.
These direct counting assays are quantitative, robust and highly tolerant of even degraded nucleic acids.
Up to 800 probes can be measured per region.
New assays from NanoString even allow for analysis of gene expression in single cells.
The NanoString platform provides a cost effective, robust and rapid technology for the multiplexed analysis of a defined set of genes, providing an excellent option for the validation of deep-sequencing or microarray based derived panels.
Please note that this pricing is for academic groups. Commercial customers should contact us for a quote.
Gene expression assays: 100 to 200 ng of total RNA is required per reaction at a concentration of 20-40 ng/µl with a total volume of 5 µl. Please inquire for more details.
miRNA expression assays: 100 to 200 ng of total RNA is required per reaction at a concentration of 33-66 ng/µl with a total volume of 3 µl. Please inquire for more details.
CNV assays: 300 or 600 ng of DNA at 100 ng/µl. Please inquire for more details.
How it works:
Each gene of interest (target sequence) has a corresponding capture probe and reporter probe. In addition to a sequence that is complementary to the target sequence, the capture probe has a biotin molecule attached (used to immobilize the sample) and the reporter probe carries a unique fluorescent barcode for detection. The reporter probes and capture probes for all genes of interest, collectively known as the CodeSet, are then hybridized to the sample. After the capture and reporter probes are bound to the mRNA, a stable tripartite structure is formed and linked to a streptavidin-coated cartridge (via the biotin capture probe) and aligned electrophoretically. The barcodes are then counted and tabulated. The Digital Analyzer uses an epi-fluorescent microscope with an oil immersion lens and a CCD camera to collect image data which is converted to a digital signal. Brightness of the code does not carry any information, the probe is simply considered present or not present. The data output is simply the gene name, accession number and number of times that transcript was counted in that sample.
A NanoString assay is a time and cost effective way to validate 40-800 unique mRNA transcripts.
While the sensitivity of a NanoString assay is comparable to qPCR, it is much more efficient.
A panel of 200 genes can be profiled in 96 samples in as little as 72 hours, using only 96 samples.
The same project performed by qPCR would require 19,200 individual PCR reactions, and would take 2 to 4 weeks to complete.
The NanoString assay is also very tolerant of material extracted from FFPE (formalin fixed paraffin embedded) tissue. The NanoString gene expression assay does not require enzymes and can tolerate both the degradation of the RNA as well as the chemical modification that tends to result from FFPE archiving.
Digital detection refers to the use of discrete units for measurement, in contrast to analogue detection, which uses the relative levels of signal (such as fluorescence) for detection and quantification. In the case of the nCounter system, the identification of each target depends only on the order of the fluors on the string (barcode) and not on the intensities of the fluors. The number of targets in a sample is quantified by counting the number of times a particular barcode is detected.
Most NanoString assays that are run by the PMGC are fully customized.
The customer works with a PMGC team member to select a panel of 20-800 different genes or target regions, which are then submitted to NanoString for the design of a custom panel or probes.
Customers will be provided with detailed information about probe design at that time.
NanoString also makes a number of catalogue and virtual panels. Information on the content of these assays can be found on the NanoString website.
The amount of material required depends on the application.
For gene expression assays, a minimum of 100 ng of total RNA or a lysate of 10,000 cells is the minimum. Total RNA, including RNA from FFPE samples, should meet minimal integrity requirements (more than 50% greater than 300 bp) in order to obtain good results.
For miRNA profiling assays, the amount of total RNA should be 200 ng per reaction.
For DNA profiling (CNV assays) 300 to 600 ng of DNA is required.
One of the biggest advantages of the NanoString technology is how well it handles degraded RNA such as that from FFPE archived materials. As a guideline, good results can be obtained if more than 50% of the sample is greater than 300 bp. To see how well NanoString works with FFPE derived RNA samples, as compared to qPCR, you can read this paper by one of our customers (Dr. Suzanne Kamel-Reid).
A unique pair of reporter and capture probes are designed for each target sequence (such as and mRNA or a potential copy number variable region of DNA). The CodeSet is the collection of all reporter probes and capture probes for all genes of interest in an assay. Off-the-shelf CodeSets (nCounter Gene Expression Panels) are available from NanoString Technologies, including the Human Kinase CodeSet and Human GPCR CodeSet. nCounter Custom CodeSets can also be made with your specific genes of interest.
As per the NanoString website, a Virtual Gene Set is “a comprehensive list of genes for the study of a genetic pathway, disease state or biological process”. The genes in each panel has been selected by mining public databases and in consultation with leading scientists in the respective fields. Virtual Gene Sets were designed to help with choosing genes to profile and provide a more rapidly available, cost effective assay. However, no matter how well curated a gene set may be, there is always a chance that your gene or genes of interest are not included. In these cases genes can be added to (or removed from) the Virtual Gene Sets. The number of genes added (or removed) will increase (or decrease) the cost of the CodeSet.
All nCounter CodeSets (both catalogue and custom) include positive control codes and synthetic RNA targets that have been spiked in to the CodeSets at concentrations ranging from 0.1 fM -100 fM, roughly corresponding to 0.2-200 copies per cell of mRNA.
A set of negative controls that should not cross hybridize to the genome/transcriptome are also included.
In the case of catalogue CodeSets, there are also a set of housekeeping genes used which can be used as controls and for normalization. It is recommended that you include 3-8 housekeeping genes in your custom CodeSets as well.
NanoString has prepared an excellent document that provides guidance on the choice of reference genes to assist in normalization. The document can be found here
Catalogue CodeSets (including miRNA) are available in 12, 24, 28, 96, 192, 384 and then multiples of 384.
The number of probes for these CodeSets is predetermined.
Custom CodeSets are available in assay numbers of 48, 96, 192, and 384, and then multiples of 384. The minimum number of probes in an assay is 20 and the maximum can be as many as 800.
A biological replicate involves independent samples (multiple patients, multiple biopsies from an individual patient, etc.).
RNA or DNA would be extracted from unique biopsies, blood from unique patients or from independent cell cultures (i.e. individual culture dishes).
The purpose of a biological replicate is to assess and control for biological diversity.
A technical replicate involves splitting a sample at some point and continuing on with the two aliquots as separate samples through the rest of the protocol. So for example, a technical replicate might involve taking one RNA sample and performing two independent amplifications and labellings from that initial sample. Similarly, if a labelled sample was split onto two arrays, that would be another type of technical replicate. Technical replicates provide an indication of measurement (or technical) error and are useful for diagnosing problems with the protocol, but offer little in the way of statistical power for a biological experiment.
The exact number of replicates required for an experiment is difficult to determine a priori without a proper power analysis. Such a power analysis is not always possible as it requires that you have an estimation of the overall variance, which you often do not have before you perform the experiment. We generally recommend doing as many biological replicates as your budget can accommodate. In general it is good to have at least three biological replicates per condition. For a more detailed determination of the number of replicates required, please contact us as we will be happy to help you design your experiment.
The nCounter™ system counts the number of targets in a portion of the sample. Rather than calculating signal intensities, whereby the intensity is proportional to the amount of target, the nCounter™ system counts the targets themselves. This system is able to detect, count and report molecules across all biological levels of expression. 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 sensitivity of the nCounter system is comparable to qPCR.
Microarray experiments are relatively complex, with numerous steps, reagents and instruments involved.
Despite this, it has been shown in many studies that the leading causes of variance are both the facility that is performing the analysis followed by the technician that completes the experiment.
All of our technicians are highly experienced and rigorously trained.
Each project is assigned a specific technician who completes the entire project to minimize variability.
Furthermore, wherever possible, we use arrays and reagents from the same lot.
We use the same equipment (hybridization oven, scanner, etc.) throughout the project; all in an effort to ensure the tightest possible data.
Often a project is large enough that it will require multiple days worth of amplification, labelling, and hybridizing to complete. In these cases we work with the customer to identify which samples are replicates and we split these replicates across the various days to ensure that it is possible to control for any day-to-day variations that may occur.
The nCounter system provides a comma separated value file that can be imported and analyzed in programs like Excel, Open Office, or Matlab. NanoString Technologies has provided guidelines for normalization and data analysis. The nCounter Data Analysis Guidelines will provide more information.