NanoString nCounter® Technology

Gene expression profiling using NanoString RNA analysis is an important step in the drug development process to determine if a therapeutic treatment is engaging with its intended target. When employed as an exploratory endpoint during Phase I clinical trials, it can help determine if the treatment is engaging its intended target and help to determine its impact on relevant pathways. NanoString RNA analysis using NanoString nCounter® technology is preferred over other techniques due to the limited number of sample processing steps needed, reducing variation.

Assay Validation

Before analyzing clinical samples using NanoString nCounter® technology, it is first necessary to validate the pre-analytical sample handling, RNA extraction, and NanoString procedures. Samples should be subjected to the same storage and shipping conditions that will be used in the clinical trial to validate the pre-analytical sample handling.

Extracting RNA for Nanostring Gene Profiling

RNA analyzed using NanoString nCounter technology on a 4200 TapeStation to assess RNA integrity.

The quality of the extracted RNA can be assessed by TapeStation to assign an RNA Integrity Number. Good quality data can be extracted from both high- and low-quality RNA samples using NanoString nCounter® technology for NanoString RNA analysis since the probes recognize a relatively short ~100 bp region of each gene, lending itself well to analysis of FFPE samples where high-quality RNA is difficult to obtain.

Gene Expression Analysis

Once the pre-analytical sample handling and RNA extraction protocols have been validated, samples can then be analyzed by NanoString nCounter® technology to assess gene expression. An overview of the data can be presented as a heatmap to identify trends in gene expression between groups and sample types.

In the example shown below, atopic dermatitis and psoriasis samples scored mild-to-moderate were used to mimic the inclusion criteria of the clinical trial. Clustering of the three different skin types indicates that each has a distinct gene expression profile. The exception to this is psoriasis sample 8, which clusters with healthy skin samples due to its low severity score. Samples from the same donor were processed using NanoString RNA analysis in parallel by two operators clustered adjacently, indicating low inter-operator variability.

Skin Punch Biopsies using NanoString nCounter Technology

NanoString RNA analysis of skin punch biopsies from (1) healthy, (2) atopic dermatitis, and (3) psoriatic donors. RNA was analyzed on a 4200 TapeStation to assess RNA integrity. RINe = RNA Integrity Number equivalent.

To reveal gene expression signatures, data can be plotted as a volcano plot. Although the samples had low severity scores, NanoString RNA analysis resulted in gene expression profiles typical of AD and psoriasis, including upregulation of anti-microbial proteins, such as the S100A proteins, lipocalin 2, β-defensin (DEFB4A) and pro-inflammatory cytokines such as CXCL8, thus highlighting the sensitivity of the technique. In a therapeutic context, gene expression data could be used to monitor PD biomarkers pre- and post-dose to assess efficacy.

Heat Map Expression Profiling

Heat map expression profile clustering of healthy, AD, and psoriasis samples (n = 3 of each) analyzed by NanoString nCounter® Technology for NanoString RNA analysis by two independent operators.

 

NanoString RNA Analysis of Skin Samples

Differential gene expression using NanoString RNA analysis of atopic dermatitis (left) and psoriasis (right) skin samples relative to healthy skin.

By assessing changes in gene expression using NanoString RNA analysis across a panel of relevant genes, biomarkers identified in preclinical research can be validated in man. Once a small panel of biomarkers is selected, these can be investigated in clinical studies. The NanoString RNA analysis approach can be used across a wide range of therapeutic areas where the relevant tissue can be accessed to allow gene expression analysis.

Find Out How

Frequently Asked Questions (FAQs) about using nCounter® technology for NanoString RNA Analysis

  • What are the advantages of using NanoString nCounter® technology for gene expression profiling?

    NanoString nCounter® technology requires very few processing steps, reducing the introduction of bias and variation as well as reducing the turnaround time. For some panels, it is also possible to simultaneously analyze both RNA and proteins in a single sample.

  • What sample types can be analyzed using nCounter® technology?

    The nCounter® technology for Nanostring RNA analysis can be used to analyze multiple biological sample types, including cultured cells, whole blood, PBMCs, plasma, serum, tissues, and even crude cell lysates. It is also well suited to analysis of FFPE tissue sections, which often yield low-quality RNA.

  • What panels are available?

    Off-the-shelf panels from NanoString are designed to broadly cover many diseases or therapeutic areas, including immuno-oncology, autoimmunity, fibrosis, CAR T cell biology, and many others. There are also panels available to detect a wide variety of the most common gene fusions and copy number variations (CNV) found in cancer and other genetic disorders. All panels can be customized with up to 30 additional genes. Alternatively, a custom panel can be built from the ground up.