What You Need to Know to Measure DNA, RNA, and Protein Simultaneously

Joseph M. Beechem on February 13, 2017

3D BiologyTechnology* Measures DNA, RNA, and Protein Simultaneously, Preserving Samples

By Joe Beechem, Senior Vice President of Research and Development

*For Research Use Only. Not for use in Diagnostic Procedures

Too often I see scientists struggling to understand their DNA, RNA, protein, and post-translationally modified protein data. They invest copious amounts of time attempting to correlate data collected on separate instruments to determine if discrepancies in their results are reflections of true biological differences or a result of error—human, instrument, analysis, or otherwise.

Researchers now more than ever seek to compare DNA, RNA, and protein levels due to the multiple levels of biological regulation occurring in key biological samples, for instance, understanding the response of the immune system to the presence of cancer and cancer immunotherapies. However, that analysis is made difficult by the disparate technologies employed for each different molecular analyte. For instance, a researcher might measure proteins using a western blot, flow cytometer, immunoassay, and then, DNA or RNA with PCR or Next-Gen Sequencing. The sample preparation, data output, and subsequent analysis for each measurement method may be completely different. This makes comparing the resultant data very difficult and leaves room for great uncertainty in scientific conclusions.

And of no less concern, with limited sample, researchers sometimes need to choose between evaluating DNA, RNA, or protein because there is not enough material for three separate measurements.

3D Biology

NanoString offers multiplexed panels that measure DNA, RNA, protein, and post-translational content simultaneously with the same detector, providing a single data output. All of this data is completely digital (i.e., quantified using single-molecule optical barcode counting), hence it can be plotted on exactly the same graph (“digital counts”). With this technology, the observed discrepancies or irregularities can be associated with true biological differences and not instrumentation and/or data normalization artifacts. We call this approach 3D Biology.

The 3D Biology panels run on our nCounter® platform, a novel technology that uses fluorescent barcodes and single-molecule counting for a completely digital fluorescence detection. Each barcode in a panel binds to a specific target in the sample (DNA, RNA, or protein) yielding a unique fluorescent signature for each analyte. The nCounter is a multiplex technology measuring as many as 800 targets in one experiment.

The approach is valuable across essentially all biological disciplines: cell-signaling, neurobiology, infectious disease, developmental biology, and cancer (to name a few). This technology is also well-suited for stem cell biology and as a monitoring technique during cell-based engineering.

Researchers can mix and match from our comprehensive library of 3D Biology panels, (at the current time focused on cancer biology and immune biology), which are available for immediate shipment. If a scientist wants to include custom mRNA or custom protein along with these multiplexed panels, that can be accomplished with about four to six weeks of lead-time.

User Experience

In addition to the sophistication of the technology, the hands-on time for experiments is minimal and complete results (data plus primary and secondary analysis) are available in about two days. The software that accompanies the instrument is especially designed for the efficient, user-friendly work-up of data that utilizes the latest understanding of biological pathways and processes. Without the need for additional software or the help of a bioinformatics expert, users can create manuscript-quality analysis and graphics.

Since the 3D Biology panels were only released starting in September 2015, work done with these panels is essentially “blue-ocean” open in terms of prior publications. Hence, almost any system measured in a 3D Biology manner is the first time DNA, RNA, protein, and protein post-translational modifications (or any combination thereof) have been measured simultaneously using a single detector with a single sample. The first peer-reviewed paper has now been published (Abey et al, BBA Clinical 7:23-35, 2016) and the AACR 2016 meeting had about ten 3D posters, abstracts, talks, and presentations.

Digital Spatial Profiling

Knowing not just which targets are expressed but also knowing where they are expressed is crucial to understanding both a tumor and the immune system’s response to that tumor.

We expanded the 3D Biology chemistry so that the 800-plex optical barcodes can also be spatially resolved. Digital Spatial Profiling (DSP) can spatially resolve both proteins and nucleic acids using just two successive 5 mm FFPE tissue slices (one for protein and protein post-translational modifications, and one for mRNA). We are currently offering a technology access program (email if interested in a project) for DSP. Up to 20 FFPE tissue slices (or tissue microarrays) can be examined in this highly multiplexed manner (multiplexed proteins are available now; enquire about select mRNAs).

In contrast to the sequential analysis of multi-target immunohistochemistry (IHC) slides, our DSP approach samples multiple markers on a single slide. This technology shortens experiments, simplifies data analysis, and provides a higher multiplexing capacity than IHC, all with the spatial context from FFPE tissue sections.

The intrinsic information content of highly multiplexed proteins and nucleic acids, all spatially resolved, makes it a great discovery platform for understanding new mechanisms in biology. This technology is truly one of a kind

At the ASCO-SITC meeting (February 23 through25 in Orlando, FL), researchers from Novartis will be presenting Digital Spatial Profiling data. Stay tuned for an upcoming post highlighting those findings.

When to Use 3D Biology

When scientists truly do not know what biomarkers they are looking for, a low-throughput survey technique like next-generation sequencing (for nucleic acids) and mass-spectrometry (for proteins) might be the best approach. But in other cases, the 3D Biology approach supported by our nCounter platform is the more efficient way to evaluate your sample for nucleic acids and proteins.

Our goal at NanoString is to create tools that help researchers capture more biology with less sample. And with our 3D Biology approach, researchers can simultaneously profile any combination of single nucleotide variation, RNA, and protein targets with as few as 5,000 cells.

If you are attending the annual AGBT meeting in Hollywood Beach, Florida (February 13 through16), we invite you to attend the presentation titled, “3D Biology view of cancer: Simultaneous detection of somatic DNA mutations and expressed fusion transcripts plus expression profiling of phospho and total signaling proteins from lung-tumor FFPE samples.” The data will be presented by Julia Kargl, Ph.D., from Fred Hutchinson Cancer Research Center. More information is available on the AGBT website.

To learn more about 3D Biology Technology, click here.

Post by Joseph M. Beechem