Gene Expression Detection Assay for Cancer Clinical Use

Narrandes, Shavira; Xu, Wayne

The nCounter technology is a robust, sensitive, reproducible, and easy-to-use method. Developed by NanoString Technologies, nCounter is a variation on the DNA Microarray. It employs two ~50 base probes per mRNA that hybridize in solution. The reporter probe carries the signal, while the capture probe allows the complex to be immobilized so that the Digital Analyzer can detect the colour codes. The Digital Analyzer senses the hybridized colour-coded probes and captures the signal intensities as gene expression mRNA levels. As the probes anneal directly to an RNA sample, no reverse transcription to produce cDNA is required. This assay has a wide range of potential applications such as in the diagnostics and validation of gene expression experiments for diseases (e.g. cancer) and research of gene regulatory pathways, ultimately leading to the translation of the data for clinical use. 36 , 37 Probe design Probe pairs are constructed for each unique gene. The probe pair is comprised of the capture and reporter probes, each 35-50 bases long and complementary to the same individual RNA molecule. The capture probe is attached via its 3' end to an affinity tag, such as biotin, while the reporter probe is detected through coupling via its 5' end to a colour-coded tag (a backbone of single-stranded DNA ligated to RNA molecules labeled with fluorophores). There are four different coloured fluorophores and six available positions on a colour code, conferring a wide range of tags that can be mixed in a single-well reaction and successfully be resolved and identified during data analysis. 36, 37, 38 Enzyme/kit As the probes attach directly to an RNA sample for its detection, no enzyme is required. The capture and reporter probes are mixed with RNA in solution prior to forming tripartite structures via hybridizing to their designated segments. Two kits of reagents are required: the CodeSet and the Master Kit. The CodeSet has customized or pre-designed probes, depending on one's experimental protocol, and a set of system controls. The Master Kit contains ready-to-load consumables and reagents for an assay. The Digital Analyzer identifies, scans, and counts the molecular barcodes per sample. 36, 37, 38 Data analysis