Digital Multiplexed Gene Expression Analysis Using the NanoString nCounter System

摘要: This unit presents the protocol for NanoString nCounter Gene Expression Assay, a robust and highly reproducible method detecting expression of up to 800 genes in single reaction with high sensitivity linearity across broad range levels. The methodology serves bridge gap between genome-wide (microarrays) targeted (real-time quantitative PCR) profiling. assay is based on direct digital detection mRNA molecules interest using target-specific, color-coded probe pairs. It does not require conversion cDNA by reverse transcription or amplification resulting PCR. Each target gene detected pair reporter capture probes carrying 35- 50-base target-specific sequences. In addition, each carries unique color code at 5' end that enables molecular barcoding interest, while all carry biotin label 3' provides handle attachment facilitate downstream detection. After solution-phase hybridization reporter-capture pairs, excess are removed probe/target complexes aligned immobilized cartridge, which then placed analyzer image acquisition data processing. Hundreds thousands codes designating targets directly imaged surface cartridge. level measured counting number times barcode detected, counts tabulated.