What is Nanostring Technology
The NanoString nCounter analysis system is the first and only platform that can deliver direct profiling of individual molecules in a highly multiplexed single reaction without any need for amplification. This protocol provides digital readouts of relative gene expression from hundreds of mRNA species simultaneously, whilst obviating any enzymatic reactions that may introduce bias in the results.
'The nCounter Platform Boasts'
• Ability to measure 20 to 800+ genes in a single reaction • Digitally detect and analyse samples in a fully automated manner • No signal amplification or polymerase steps required • High flexibility in sample input (including mRNA, miRNA, FFPE samples and whole cell lysate) • High throughput with minimal hands-on time
'How the nCounter Analysis System Works'
For every gene sequence to be interrogated, two target specific probes are created complementary to the mRNA sequence selected for the gene of interest. One carries a sequence of fluorescent tags that are used as a molecular barcode for direct digital detection, while the other carries a biotin molecule used for immobilisation of the sample cartridge during the process of data acquisition. The fluorescent-tagged 'Reporter Probe' is assigned a specific sequence of 7 tags allowing for thousands of barcode combinations. The two probes are provided in excess in order to drive the hybridisation reaction to completion, with any unbound or partially bound probes removed through a series of automated washing steps performed by the NanoString Prep Station prior to the detection and analysis process.
After removal of excess probes, the captured mRNA is bound to the streptavidin-coated cartridge surface by its bound biotin probe. At this point an electrical current is used to align the mRNA-probe complexes, which are then immobilised using a proprietary reagent. Once immobilised the sample cartridge is stable and can be stored at 4 degrees Celsius for some time if necessary.
For analysis, the cartridge is transferred to the Digital Analyser where an automated optical camera records up to 555 fields of view across the cartridge, simultaneously detecting and recording the number of sequence specific probes bound to the surface of each sample cell. As the platform is directly imaging individual molecules, we are able to count the actual number of times each code is found in the sample.
These counts are provided as a simple RCC file for use in excel, 'R' or NanoString's own analysis and normalisation software 'nSolver'. To ensure validity, the sample aliquots contain a number of pre-defined positive and negative controls as well as a number of 'housekeeping' gene target probes which are counted along with the gene sequences being investigated for simple standardisation of sample counts.