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nCounter® and RNA Gene Signature Development

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1. What is RNA signature development?

RNA gene signature development is the process by which you take a discovery experiment on thousands of genes and you reduce that gene expression measurement down to dozens of genes where the pattern of gene expression, in those dozens of genes, is reflective of a biological phenotype in the experiment.

2. Why is it important?

It's important because cancer is complex, and we need to distill all of that biology down into a much simpler measurement. Now, RNA expression signatures are easier to measure than proteins, proteins are just too labile to measure. On the other hand, you can measure all of the genotypes in a tumor, but that information is not deterministic.

3. What are the biggest problems?

The biggest problem in developing an RNA expression signature is running lots of samples. Not just any samples, in oncology, you need to be able to run FFPE samples. Not just any FFPE samples, old age FFPE samples that have matured clinical data associated with them. That's essential to develop a good expression signature. So now when you're running those samples, you're actually making a transition from fresh frozen tissue where all these discoveries were made, to FFPE tissue where you're going to develop the signature, and then validate that signature. So it's the proverbial, “building the ship while you're sailing it”. You're actually trying to reduce these genes while at the same time, you're transitioning from one platform to another; transitioning from one tissue type to another tissue type. That's very scary as an assay developer because you may actually throw out genes that contain valuable information.

4. How do scientists deal with these problems?

Scientists traditionally deal with these problems using quantitative PCR (qPCR). So what you do with quantitative PCR is you have to optimize each and every one of your primers for old age FFPE samples. And that can be very difficult to do. That can take, in some cases, years to optimize those primers. Then, once you've actually got optimized primers, then you need to optimize your process across all the different genes and you need to maintain that optimization, maintain that quality over long periods of time; during which you're running your validation studies, even into the clinic, where you are going to be testing patients.

5. How does nCounter® solve these problems?

So as an assay developer, my biggest fear is when I'm developing an RNA expression signature that actually throws out genes that are of high value. So the nCounter System solves this problem by allowing you to put in hundreds of genes into that experiment and actually reduce to the optimal set of genes, starting with a wide net. So that solves the problem of optimizing the signature itself. That's all done in a single tube, without any enzymology. That means you don't actually have to go through and optimize every single one of your primers for old age FFPE samples. This can all be done in a single tube, in a very short period of time.

6. What is the best example of this?

A good example of this comes from a group of lymphoma researchers who discovered an RNA expression signature about a decade ago and have been researching that signature for this entire time. It was only when the nCounter System came along when they could do the experiment that they needed to do. They've owned a CodeSet of 800 genes, so a large CodeSet, they ran that on a bunch of FFPE samples to reduce that down to only 20 genes and validate that signature for use in the clinic, in only about a year.