Researchers who routinely perform gene expression monitoring desire greater sensitivity, accuracy and reproducibility coupled with a high capacity for looking at many genes simultaneously. NanoString's nCounter™ Analysis System represents the only platform capable of highly multiplexed direct digital quantification of individual target mRNA molecules in a biological sample without the use of enzymes or amplification which can introduce bias to the data.

Multiplexing

The nCounter™ Analysis System is capable of measuring up to 1000 genes simultaneously in a single tube. In the experiment graphed below, the expression level of 500 genes was determined for two different cell populations with the same sensitivity as one would get using RT-PCR.

Sensitivity

Sensitivity is extremely important for most biological research. The vast majority of genes expressed in a given tissue are expressed at 10's of copies per cell or less. NanoString reporter technology has shown sensitivity at or below a single copy of mRNA per cell even when measuring hundreds of genes simultaneously.

In this chart we demonstrate the ability of NanoString reporters to detect, with statistical significance, target molecules spiked into total RNA down to a level of 100 attomolar. This represents a concentration that is significantly less than a single copy per average cell. It is also important to note how linear the measurements are over a 1000-fold dynamic range. Although we believe that the dynamic range could go much higher, because we do not require amplification of any kind, this is more than sufficient to cover the vast majority of genes of interest.

Accuracy

Because there are no enzymatic reactions required to prepare the sample, we do not introduce bias into the data that are artifacts of reverse transcription or PCR amplification. We simply measure the RNA abundance in a sample by tagging and counting individual mRNA molecules.

Precision

Precision, or reproducibility, is also very important when performing gene expression measurements. High precision measurements build statistical confidence into the measurement and help eliminate random noise.

The overall precision of the technology has been shown to be very good. We performed triplicate measurements of more than 100 endogenous genes in commercially available human placental total RNA. We measured expression levels with consistently tight CV's across a broad range of relative abundance levels.

Using the spikes from above to establish the relative abundance of each transcript measured, the %CV was less than 20% all the way down to a single copy per cell of sensitivity.

Sample Size

When individual molecules of targets can be counted, it dramatically reduces the amount of biological sample required for analysis. Amplification is required by most other technologies in order to generate a signal sufficiently above background. In fact, with small sample quantities, two rounds of amplification are often required for microarray analysis which can even further bias the data.

With NanoString technology, each reporter probe is hybridized to it's mRNA target and forms a single molecule that we image and count. Today we routinely work with 100ng of total RNA and have demonstrated good results at 10ng. We expect to be routinely working at 10ng in the near future.

Ease of Use

Our gene expression assay is very simple and straightforward. The are no complex protocols to reverse transcribe the mRNA. Additionally, there are no cDNA synthesis or amplification steps. With simple, easy to perform protocols for target preparation there is less chance of error and lower variability overall leading to a more robust gene expression measurement system.

Time to Result

Since the hybridization reaction is in total RNA, we eliminate many time consuming protocols and shave days off the overall time to result.