March 15, 2016 (Vol. 36, No. 6)

Xiao-Jun Ma Ph.D. Chief Scientific Officer ACD

A Guide to Investigating Noncoding RNA Species

The significance of noncoding RNA is becoming increasingly recognized as new classes involved in genetic regulatory control and diverse cellular activities are being uncovered on a regular basis. Discovering the world of noncoding RNAs has already unlocked many secrets about how life’s incredible phenotypic diversity arises from a relatively small and fixed set of protein-coding genes. 

Moreover, transcriptomic profiling has also proven that, like protein, RNA is a rich source of biomarkers. Having long been used for diagnostic testing and the identification of potential therapeutic targets, biomarkers are growing exponentially in their importance within the era of personalized medicine. In fact, several noncoding RNA candidates have shown promising diagnostic and prognostic utility (Figure 1).

To fully capitalize on the properties of noncoding RNA, it is crucial that effective analytical technologies are employed, and this presents a challenge. For protein coding genes, immunohistochemistry is used routinely to map gene expression to specific cells in situ, yet noncoding RNA species lack protein counterparts. For these molecules, gene expression can only be visualized by RNA in situ hybridization (ISH).

This tutorial describes a modern approach to investigating noncoding RNA for advancing studies in a range of areas from cancer research to neuroscience.

Figure 1. RNA ISH in prostate tumor tissue. This whole-tissue section was probed for the noncoding PCA3 transcript using RNAscope ISH technology.

From Discovery to Understanding

In order to interrogate the transcriptome for noncoding RNA species, a fully comprehensive transcriptomic discovery program will involve utilizing technologies such as the microarray or RNA-Seq. Once a set of transcripts exhibiting differential expression has been identified, the investigation must then focus in on validating their functional significance and biological or clinical relevance. When it comes to studying those transcripts of interest in greater depth, RNA ISH provides information regarding the expression of noncoding RNA species within the morphological context, which is vital for understanding underlying biological mechanisms.

RNA in Situ Hybridization

The spatial resolution provided by RNA ISH presents a critical data dimension in gene expression analysis, for example providing precise localization of target RNA within a cell, or allowing direct mapping of RNA biomarkers to specific cell types in tumor tissue (e.g., stromal versus tumor expression). However, in the past this technique suffered from poor sensitivity, and only with ongoing technological advances has the performance and ease of use improved sufficiently to make RNA ISH both widely established and accessible.

Researchers now have access to several innovative approaches, which have reinvented the traditional technique to offer improved sensitivity and signal-to-noise ratio. These include RNAscope® ISH technology, based on ACD’s (Advanced Cell Diagnostics) probe design and signal amplification system. In order to substantially increase the signal-to-noise ratio, two independent probes (double Z probe pairs) must hybridize to the target sequence in tandem for signal amplification. It is highly unlikely that two independent probes will hybridize to neighboring nonspecific sites, so this design concept ensures selective amplification of target-specific signals while preventing amplification from nonspecific binding.

For the analysis of noncoding RNA species, which tend to have lower expression levels, the advent of modern RNA ISH is especially relevant, as the highest detection sensitivities are required. Employing such methods is therefore key to single-cell gene expression analysis of noncoding RNA in situ with single-molecule sensitivity, enabling both biological insights and biomarker applications (Figure 2). Providing an example of how to utilize such a technology, the following case study looks at developing cancer biomarkers.

Figure 2. Visualizing noncoding RNA. Expression of HOTAIR RNA (brown dots) in human breast cancer FFPE tissue was assessed via RNA ISH using RNAscope® 2.0 HD Reagent Kit-BROWN.

Case Study: Cancer Biomarkers

Researcher Rohit Mehra, M.D., is clinical assistant professor of pathology at Michigan Center for Translational Pathology, and has employed RNAscope technology as part of his work in utilizing long noncoding RNA (lncRNA) as a cancer biomarker. This type of noncoding RNA plays an important role in the pathogenesis of genitourinary cancers, especially prostate cancer. Being a relatively new field of study, a full understanding of their role in normal and disease states has not yet been reached. Of the several lncRNAs that are known to be significant in prostate cancer, one in particular—SChLAP1—may have clinical utility as a prognostic or diagnostic biomarker. Accessible methods for routine in situ lncRNA detection are vital for investigating this, and the overall methodology is formed of three separate stages, described as follows:

  1. Transcript Discovery: Comprehensively profiling the transcriptome of >100 prostate cancer tissues and cell lines using RNA-Seq found that ~20% of RNA transcripts in prostate cancer represent novel, uncharacterized lncRNA genes. From this set, 121 candidate lncRNAs were nominated for further investigation.
  2. Biomarker Validation: One of these was re-named SChLAP1, and in the cohort studied, RNA ISH with RNAscope technology effectively stratified patient outcomes by predicting more rapid biochemical recurrence, clinical progression to metastatic disease (defined by a positive bone scan), and prostate cancer–specific mortality.
  3. Functional Investigations: Dr. Mehra’s early studies, which involved the use of RNAscope technology, found that SChLAP1 antagonizes the genome-wide localization and regulatory functions of the SWI/SNF chromatin modifying complex. Furthermore, through antagonizing the tumor-suppressive functions of the SWI/SNF complex, it contributes at least in part to the development of lethal cancer. Further studies to uncover the molecular mechanisms by which SChLAP1 functions are ongoing.

The application of modern RNA ISH technology in Dr. Mehra’s work allowed direct visualization of gene expression in the target tissue of interest: The same sample could be analyzed to investigate whether gene overexpression occurs in benign prostate glands, high grade prostatic intraepithelial neoplasia (HGPIN, a precancerous state), or prostate cancer.

One important aspect of this study is the ability of the RNA ISH technology to detect single RNA molecules in routine formalin-fixed, paraffin-embedded (FFPE) clinical specimens, which was previously a hurdle to applying RNA ISH in the translational medicine setting.

This study has shown how, thanks to technological developments, RNA ISH now allows for effective and reproducible in situ detection of noncoding RNA, opening up the use of lncRNA as a biomarker. 


Major advances over the last few years have changed the status quo, transforming the capabilities of RNA ISH. It is now possible for researchers to detect noncoding RNA with single-molecule sensitivity in a range of sample types, including FFPE tissue, while preserving tissue context with single-cell resolution. A number of innovative research studies like the one discussed have already capitalized on these capabilities, and this technology can be applied to a range of study areas. Thus, modern approaches to RNA ISH, such as RNAscope technology, now serve to fill the long-standing gap of in situ detection of noncoding RNA throughout life science and clinical research.

Xiao-Jun Ma, Ph.D. (, is CSO at ACD.

This site uses Akismet to reduce spam. Learn how your comment data is processed.