NanoString nCounter^TM System

Each gene of interest requires a corresponding capture probe and reporter probe. The reporter and capture probes for all genes of interest are collectively known as the Code Set. The first probe, or capture probe, contains a 35-50 base pair sequence complementary to a target mRNA with a short common sequence coupled to an affinity tag, such as biotin. This capture probe, specifically the affinity tag, allows the complex to be immobilized on the cartridge for data collection. The second probe, or reporter probe, contains a second 35-50 base pair sequence complementary to the target mRNA, which is coupled to a color-coded (fluorescent) tag that provides the detection signal. The probes are hybridized directly to one of several targets (i.e., RNA, miRNA or DNA), where a stable tripartite structure (target/capture/reporter) is formed and linked to a streptavidin-coated cartridge. With the fixed structure, the fluorescent tags can be counted for data analysis.

The Digital Analyzer counts the number of fluorescent tags bound. The Digital Analyzer utilizes an epi-fluorescent microscope with a high magnification lens and CCD camera to collect image data (See Figure 1) and then converts it to a digital signal.

The workflow is as follows:

Service includes:

• Consultation for reagent ordering (reagent price list HERE) and protocol development
• Assistance with experimental design
• Bioanalysis of input material for accurate quantitation and RIN
• Preparation and processing of 12 RNA or DNA samples per cartridge, hybridization and imaging of cartridge
• Output in CSV file and nCounter advanced analysis

The price of service depends on the scope of the work. Please contact us (pamela.plant@unityhealth.to) for more information.

Reagent pricing: Reagents are purchased directly through Nanostring. Click here for reagent price list or contact info@nanostring.com

Sample requirements:

• Samples can be extracted from fresh or frozen tissue, cell lines, FFPE samples. Total RNA, miRNA, DNA or crude lysate can be used depending on assay type
• mRNA assays minimum of 100 ng total RNA (minimum concentration 20 ng/ul)
• miRNA assays – between 100-300 ng total (minimum concentration 33 ng/ul)
• CNV or ChIP-String assays – 200 – 600 ng DNA (minimum 29 ng/ul)

• It allows the direct measurement of mRNA expression levels without the use of enzymatic reactions
• NanoString can analyze crude preparations including fragmented RNA. Is an excellent validation tool for microarray and RNA-Seq data (methods which typically require high-quality RNA)
• Using small quantities of RNA is both time-effective and cost-effective
• Offers a high sensitivity and a high multiplex capacity (500+ genes), allowing for accurate quantitation across numerous genes
• NanoString provides a digital readout. This reading is linear across a large dynamic range, reduces background noise, and allows for more accurate downstream analysis
• Simple data output is delivered in a CSV file

• All are techniques used for differential gene expression studies but not all are discovery based platforms
• Microarrays detect nucleic acids in a sample by hybridization to spotted probes on microchips. Microarrays are useful for enabling a high degree of multiplexing (assessing thousands of genes simultaneously) and rapidly assessing multiple samples. Background hybridization and probe saturation are the main problems that interfere with sensitivity and detection. It is often used as a discovery based platform but since microarrays detect only known sequences, so they can’t be used for true discovery (of unknown sequences) like RNA-Seq
• RNA-Seq is optimal for discovery based research as the technique is not limited to profiling known genes. However, RNA-seq generates a staggering amount of data so downstream post-sequencing analysis remains complex. Post-sequencing analysis of short-read RNA-seq is also complicated by RNA fragmentation. The sequence reads don’t necessarily tell you what the full-length RNA looked like
• NanoString is not for discovery but is optimal for targeted transcriptomics. The advantage over NGS is there’s no library to make, no enzymes, no processing to introduce error and bias. There is a high degree of multiplexing (albeit not as high as microarray and NGS) and sensitivity as well
• Quantitative real time PCR or qPCR is for targeted differential gene expression only. It is highly sensitive but very limited as only one gene is targeted per sample
• Here are publications comparing Nanostring with microarray (Richard et al., 2014) and qPCR (Prokopec et al., 2013)

The prep station will process 12 samples (per cartridge) at any given time. 6 cartridges can be put in the nCounter (counting) station. Pre-fabricated Code Sets can be purchased in quantities of 12, 24, 48, 96, 192 and 384 (and even higher in multiples of 384).

All custom and pre-fabricated nCounter code sets include positive controls and synthetic RNA targets that have been spiked in at a range of concentrations for Quality Control (assessing the success of the run). Negative controls (sequences that should not cross hybridize to the genome/ transcriptome) are also included. Housekeeping genes are included in pre-fabricated code sets that can be used for normalization but these have to be designed into custom sets.

• PlexSet offers the user more flexibility in probe design. The technology is identical but the reporter and capture probes are not directly complexed to the target specific primers. Instead, reporter and capture tags have unique sequences which are designed into the (compatible) forward and reverse target specific primers by the user (see Figure below). Targets in Plex-Set assays are counted in an identical way to assays using pre-fabricated and custom code sets but instead of a tripartite complex (with target/Reporter/Capture), the PlexSet forms a quaternary complex
• Click here for a visual representation of PlexSet

Nanostring offers routine and advanced analysis modules which run in the nSolver software. You can also choose to export the data (digital counts) from an nCounter experiment to a CSV file, which can be imported into third party analysis program (i.e. Gene Pattern, Broad Institute or Matlab etc.). Data analysis manuals are available for all applications on the Nanostring website and are excellent resources for guidelines on normalization and data analysis.