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Columns: March 15, 2017 (Vol. 37, No. 6)

Singling Out RNA Biomarkers in Situ

ACD’s ISH Platform Achieves Single-Base Resolution and Retains Morphological Context

  • Advanced Cell Diagnostics (ACD), a company recently acquired by Bio-Techne, has introduced assays that promise to transform molecular pathology. These assays can detect and quantify RNA in situ while providing single-molecule sensitivity and single-base discrimination. Such sensitivity and specificity is unprecedented. Moreover, ACD technology is compatible with automated tissue-staining systems and pathology laboratory workflows.

    ACD technologies include RNAscope, an in situ hybridization (ISH) assay platform, and the BaseScope™ product, an assay for the detection of short targets and exon junctions. Together, RNAscope and BaseScope help researchers detect small differences in RNA transcripts of expressed genes at single-base resolution.

    These technologies can be used to detect long noncoding RNAs at a level of detail that detects alternative splice junctions, point mutations, small insertions or deletions, and other minute differences in sequences directly, within the full context of tissue and cellular morphology. Essentially, they not only capture gene expression, they also allow it to be visualized within intact tissue.

    “This is truly unprecedented in the industry,” asserts Yuling Luo, Ph.D., ACD’s founder, president, and CEO. “Before BaseScope was introduced last fall, people could detect mutations or single-nucleotide variants only by lysing the tissue, isolating the DNA or RNA, amplifying specific sequences, and then looking for mutations through sequencing or PCR.

    “BaseScope, for the first time, allows one to determine whether mutations and other genetic alterations are present as well as their distribution throughout the tissue and the cells in which they occur. This ability to visualize subtle genetic changes in the morphologic context is something no one thought was possible.”

  • BaseScope Capabilities

    The technology operates with sequences as short as 50 bases. Unlike the “grind-and-bind” bulk cell population analyses associated with traditional gene-amplification methods, such as quantitative PCR (qPCR), BaseScope can accurately localize the exact cells in which the genetic alterations are present.

    It is also faster. Rather than waiting one to two weeks for specialty laboratories to generate results from traditional quantification methods, scientists can use BaseScope assays to run samples in their own laboratories, and do so just as easily as they might run fluorescence in situ hybridization (FISH) and other immunohistochemistry assays. Then, the scientists may interpret the results and visualize the expression of molecular markers within their routine workflows.

    “They can have results in one to two days,” Dr. Luo says. And, unlike qPCR and next-generation sequencing, the morphological integrity of the tissue remains intact so sample heterogeneity also can be evaluated.

    Since BaseScope assays were launched in 2016, they have been used to facilitate CRISPR/Cas9 gene editing, detect splice variants, confirm gene knock-outs or knock-ins, and identify antigen-specific T-cell clones in immuno-oncology. “These are all things people couldn’t do before,” Dr. Luo states.

    By understanding the basic mechanisms, pathologists and drug developers can work more accurately, detecting and validating targets that are expressed at low levels, which is especially important in immuno-oncology. “This change lets researchers take ownership of the results and to interpret molecular markers within the context of histology. That’s never been possible before,” Dr. Luo reiterates.

  • RNA ISH Advances

    The speed of assay development was notable most recently in the development of assays for the Zika virus. “RNAscope Zika assays already have been used in seven high-impact clinical research papers by leading Zika researchers—including the CDC—to understand Zika-associated pathogenesis,” notes Dr. Luo.

    Today, RNAscope technology is automated on the Leica Bond and Roche Ventana Discovery series of instruments for formalin-fixed paraffin-embedded (FFPE) tissue. For users focused on downstream data analysis—an area that is attracting more attention—specialized third-party tools such as Indica Labs’ Halo software or Leica’s Aperio RNA ISH algorithms are available. Such tools can extract quantitative information from RNAscope-generated images.

    At present, most assay analysis in this segment of the industry is manual. Although the complexities that attend manual approaches may be tolerable in research applications, they are hardly sustainable in clinical diagnostics. “With manual systems, scientists must look at images and visually estimate levels of gene expression,” Dr. Luo elaborates. “It is very tedious and leads to different results by different persons.”

    Automation eliminates human subjectivity, to deliver consistently accurate results and to ease assays toward clinical use. “Automation workflow,” Dr. Luo contends, “is one of the most important aspects for diagnostics.”

  • A Bio-Techne Brand

    Bio-Techne’s acquisition of ACD, which occurred in the summer 2016, was intended to expand the purchaser’s presence in clinical laboratories through genomics solutions. “There’s been a very smooth transition,” notes Dr. Luo. “BioTechne is very supportive of what we do.” Dr. Luo adds that the strategic fit between the two companies is excellent. For customers at clinical laboratories, the combined company provides a one-stop shop.

    ACD’s product line includes more than 13,000 probe reagents and kits for automated and manual uses. In 2016, the company introduced four new products and is currently working on a high-multiplex assay. “We try,” states Dr. Luo, “to anticipate the challenges our customers will encounter as they learn more and more about the molecular foundations of biology and disease.”

  • Advanced Cell Diagnostics (ACD)

    Location: 7707 Gateway Boulevard, Newark, CA 94560

    Phone: (510) 576-8800

    Website: acdbio.com

    Principal: Yuling Luo, Ph.D., Founder, President, and CEO

    Number of Employees: 100+

    Focus: ACD develops cell- and tissue-based gene-expression assays for molecular pathology in academic research, drug discovery, development, and companion diagnostics.