Scenario description
In the heterogeneous target expression scenario cells display different levels of staining for the target RNA, indicating heterogeneous gene expression among the same cell type (Figures 3 and 4). In this scenario both the expression level and the percent of cells expressing the target at different levels could be of importance.
Figure 3. Heterogeneous expression for AFAP1-AS1 (A) and positive control staining (B) in human lung cancer tissue using the RNAscope ® 2.5 HD RED assay. Shown are two foci, one that expresses AFAP1-AS1 (white circle) and one that does not express AFAP1-AS1 (black circle)(A). This demonstrates heterogeneous AFAP1-AS1 expression between two tumor foci and suggests the presence of two clonal tumor populations. The expression of the PPIB housekeeping gene is uniform between the tumor cells of each foci, indicating good quality RNA expression in both foci (B).
Figure 4. Heterogeneous expression of PD-L1 in green showing high, medium and low expression in human lung cancer tissue using the RNAscope ® 2.5 HD Duplex assay. CTLA4 is shown in red.
Analysis guidelines
The expression level and the percent of cells expressing the target at different levels can be analyzed in the following ways:
Assess the overall expression level of the entire sample (average number of dots per cell) using methodology #1 or methodology #2.
The dynamic range of expression (cell-by-cell expression profiles) can be quantified for the entire tissue section or selected regions of interest by binning cells with different levels of expression into separate bins as shown in Figure 5. The data can be presented as a histogram to represent the expression level distribution or can be calculated as a Histo score (H score). This H score can be achieved by semi-quantitative analysis or quantitative image- based software analysis where the percentage of cells within each bin characterized by a certain expression level or number of dots (ACD scores ranging from 0 to 4) is estimated and the overall H score (range of 0 to 400) is calculated as follows:
