nCounter® Single Cell Gene Expression Assay
Make Every Cell Count
nCounter® Single Cell Gene Expression Assays offer an exceptional approach to identifying cell-to-cell differences within a population of cells. Each highly multiplexed, single tube assay allows the analysis of 20 – 800 genes and frees you from the constraints of fixed format microfluidic platforms. Let biology guide your research.
- Analyze multiple pathways for up to 800 genes
- No need to match gene number to a fixed format consumable
- Obtain single cell sensitivity while minimizing amplification
- Single molecule counting minimizes data noise
VIDEO: Dr. Joe Beechem on Single Cell Digital Gene Expression with the nCounter Analysis System
The introduction of the nCounter® Single Cell Gene Expression Assay greatly expands the types of studies that can be analyzed on the nCounter Analysis System, allowing researchers to address biological questions that have previously been impossible to answer. Entire biological pathways and custom gene signatures can now be studied for single cells without the necessity to match the gene number to the format of a microfluidic PCR consumable.
The Power of Single Cell Analysis
Historically, gene expression profiling has been performed on populations of cells, where observed expression levels represent an amalgam of the unique expression states of each cell within the population. The recent emergence of single cell RNA analysis using qPCR has provided an avenue to discern new mechanistic insights within individual cells and a means of elucidating previously hidden relationships between individual cells within a population. Whilst these new tools have great utility, the constraints of the associated fixed format consumables, means that they are limited in their ability to support the accelerating study of pathway- based biology.
In order to demonstrate how analyzing single cells can uncover significant differences in gene expression profiles that are hidden when looking at larger cell populations, individual (and small populations) of CD8+ T-cells were flow-sorted. After reverse transcription and amplification using the nCounter Single Cell Gene Expression Protocol, the gene expression profile for each sample was generated using a 191 gene custom CodeSet on the nCounter Analysis System. The data clearly demonstrates that individual single cells have very different expression profiles, however this diversity is masked in the samples containing 10 pooled cells, and almost completely hidden in the samples containing 100 pooled cells.
High Sensitivity with Linear Response
The nCounter Single Cell Gene Expression Assay provides an ultra-sensitive, reproducible, and highly multiplexed solution for profiling gene expression from single cells or as little as 10pg of total RNA. A Multiplexed Target Enrichment (MTE) step allows transcripts within individual cells to be linearly amplified after a reverse transcription step.
A Multiplexed Target Enrichment (MTE) was performed with 800 primer pairs using 100 pg of either Human Reference or Brain Reference total RNA as sample input. Fold changes were calculated for all probes exhibiting significant detection and plotted against the fold changes observed in unamplified samples (100ng) for the same genes after hybridization with nCounter GX probes. These data show a 1:1 correlation of fold changes between assays, demonstrating the simultaneous, unbiased amplification of hundreds of target transcripts and preservation of fold change information provided by the nCouner Single Cell Gene Expression protocol.
nCounter Single Cell Gene Expression Assay Workflow
Single Cell → Single Tube → 800 Genes (click image below for larger view)