RNAscope Assay Visualization and Interpretation

Please contact Indica Labs directly at info@indicalab.com for HALO related questions.

You can analyze your image either semi-quantitatively or quantitatively. Please refer to the scoring guideline for semi-quantitative analysis or use image analysis software to analyze quantitatively. ACD has technical notes providing guidance on how you can use ImageJ/Cell Profiler/QuPath to quantify gene expression from your RNAscope images. In-house ACD uses HALO software from Indica Lab for quantitative analysis.

ACD always recommends running 3 slides minimum per sample: your target marker panel, a positive control, and a negative control probe.  The positive control will help determine whether the quality of RNA in the tissue specimen is sufficient for detecting your RNA target. The bacterial dapB negative control will help determine whether the tissue specimen is appropriately prepared for RNAscope and BaseScope assays.

The RNAscope Multiplex Fluorescent v2 Assay and HiPlex v2 Assay can be imaged using either an epi-fluorescent or confocal microscope with the appropriate filters for assigned fluorophores. For specific excitation wavelength of each probe channel please refer to the appropriate user manual for your assay of interest.

RNAscope signal is detected as punctate dots. Clusters can result from overlapping signals from multiple mRNA molecules that are in close proximity to each other.

There may be variation in dot intensity/size which is because of the differences in the number of ZZ probes bound to a target molecule. However, since each dot represents a single transcript, the number of dots is critical and not the intensity/size of the dot(s).

RNAscope signal is shown as punctate dots. Each dot represents a single copy of an mRNA molecule.