To accurately interpret gene expression in tissues, scientists are in need of a quantitative assay that provides spatial information at the molecular level. The RNAscope™, BaseScope™, and miRNAscope™ ISH technologies enable single molecule detection of RNAs in situ within the tissue context. Our highly trained scientific experts and pathologists offer various quantification methodologies. The analyses are defined according to the biological question being addressed, and each project is supported by consultations with the scientific team.
• Quantitative cell-by-cell gene expression data
• Identify tissue types or regions of interest
• Optional heat maps provide full-tissue spatial expression
Image analysis is performed using HALOTM (Indica Labs), the powerful digital pathology tool for quantitative gene expression analysis. HALO reports morphological and multiplexed gene expression data on a cell-by-cell basis across entire tissue sections, and is applicable for RNAscope, BaseScope, and miRNAscope singleplex, duplex, and multiplex fluorescence assays. Sorting and filtering capabilities allow the user to mine millions of cells while visually assessing the corresponding cells in situ. The software can be trained to classify cell populations or regions of interest (ROI), for example categorizing a tissue into tumor cells versus stromal cells.
Heterozygous expression of EGFR in human breast cancer tissue. RNAscope staining (left) and HALO image analysis markup (right), showing cells binned according to level of gene expression based on dots per cell
Example of HALO analysis of NSCLC tissue stained with the RNAscope Duplex assay for CD45 (red) and PD-L1 (green). Co-expressing cells shown in yellow
PATHOLOGIST REVIEW
• In-house pathologists for image annotations
• Histopathology notes include tumor load and visual H-scoring
• Teleconference with pathologists for data presentation and review
Your pathologist’s workload can be reduced by utilizing our team’s in-house and contract pathologists who will provide additional notations or review for any study. Annotations can be performed manually either to identify regions of inclusion or regions of exclusion, for instance to exclude regions of necrosis or poor positive control staining. Visual H-scoring can also be performed by a pathologist to provide more quantitative results (see manually annotated regions of interest in tumor tissue sample by our pathologist below).
Tumor compartment only
Tumor-associated stroma
Tumor and tumor-associated stroma
Semi-Quantitative Visual Scoring
•Semi-quantitative scoring performed by highly trained experts
•Rapid scoring shortens turnaround time and speeds delivery of results
•Provides expression level and percentage of cells positive
For gene expression studies where understanding relative signal intensity is sufficient, visual semi-quantitative scoring can be used to provide a single score based on the expression level observed. RNAscope ISH staining generates punctate chromogenic or fluorescent signals. Scoring is based on counting the number of dots per cell, with each dot corresponding to a single RNA molecule. Visual scoring (0–4) is performed based on the predominant staining pattern seen throughout the entire sample or within a defined region of interest. Heterogeneity or non-uniformity of expression will be noted. Percentage of cells positive is scored visually based on number of cells with ≥1 dot/cell and binned into categories (i.e., 0%, 1–25%, 26–50%, 51–75, 76–99%, 100%).
Scores (0–4) are assigned to a tissue sample based on the number of dots/cell
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