nCounter® PanCancer Immune Profiling Panel (NanoString Technologies, Inc., Seattle, WA)
© Cesano. 2015
Received: 11 August 2015
Accepted: 15 August 2015
Published: 15 December 2015
Hybridization: unique pairs of a “capture” and a “reporter” probe are provided for each gene of interest, allowing up to 800 genes to be multiplexed, and their mRNA transcript levels measured, in a single experiment, for each sample. The “reporter” probe carries the signal, and the “capture” probe allows the complex to be immobilized for data collection.
Purification and immobilization: after hybridization, samples are transferred to the nCounter Prep Station where excess probes are removed and probe/target complexes are bound, immobilized, and aligned on the nCounter Cartridge.
Counting and Analysis: sample cartridges are placed in the nCounter Digital Analyzer for data collection.
The time from sample lysates to data results is two days and because the process is highly automated the hands-on time (and therefore room for human errors) is limited (25 min per 12 samples). Measurements are performed using the commercially available nCounter Analysis Instrumentation at the site of sample collection or through working with any of the multiple Contract Research Organizations offering NanoString services.
Identifying 24 different infiltrating immune cell types, such as those in a peripheral blood mononuclear cells (PBMC) population or infiltrating into a tumor.
Assessing immunological function and response to immunotherapy, such as immune checkpoint regulation.
Identifying tumor-specific antigens, such as cancer-testis (CT) antigens.
Housekeeping genes that facilitate sample-to-sample normalization.
Type of data obtained/readout
Barcodes are counted and tabulated for each target by the nCounter Digital Analyzer. The data readouts are either the composite of the individual expression profile of cells within that population; or detailed, single-cell level expression, which may represent biologically relevant small percentage (5-10 %) of the entire population of the cells. The instrument analysis software automatically performs QC, normalization, data analysis and creates multi-page reports with the options of performing advanced analyses including pathway applications.
Limitations of the approach
The data are not spatially resolved, hence, it represents the average of a few 1000’s of cells
The data are targeted discovery (as contrasted with pure discovery), and measure only the 770 genes predefined in the panel. It is possible to add 30 completely custom genes to the 770-plex panel (total of 800-plex)
While it is possible to resolve alternate splice transcripts and expressed gene-fusions, the panel does not measure single-nucleotide polymorphisms (SNPs).
Advantages of the approach
Multiplex hundreds of gene targets in a single reaction
High sensitivity (<1 copy per cell)
No enzymes or amplification required to perform assay, ideally suited for FFPE samples and cell lysates
Multiplex 800 regions from as little as 25-300 ng of total RNA
Completely digital detection (all quantitation is by direct single-molecule counting)
Automated analysis software
Types of samples needed and special issues pertaining to samples
The nCounter PanCancer Immune profiling panel is fully compatible with clinically relevant sample types such as fresh-frozen (FF) tissue, formalin-fixed paraffin-embedded (FFPE) tumor sections, isolated immune cell populations such as PBMC and cell lysates. For very low input samples (even down to single-cell work, such at CTCs), a multiplexed target enrichment amplification protocol is available.
Level of evidence
There have been bout over 800 peer-reviewed publications using the nCounter Analysis System. The instrument, reagents and software have received 510(k) clearance from the FDA for use with the Prosigna Breast Cancer Prognostic Gene Signature Assay . There is a growing body of literature available demonstrating the use of the nCounter analysis system using much of the same content as in the nCounter PanCancer Immune Profiling Panel in immuno-oncology setting .
Cancer testis antigens
Circulating tumor cells
Food and Drug Administration
Peripheral blood mononuclear cells
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