Image Analysis

RNAscope™ and BaseScope™ assays result in signals where mRNA molecules are represented in punctate dots. These signals can be acquired by fluorescent or chromogenic detection modalities with appropriate image acquisition process.

Assay selection guidelines

Start with the selection of the right assay based on your samples/study. Please follow the guidelines in the following link:

  • For chromogenic assays, any standard brightfield microscope or digital slide scanners can be used to acquire images at 20x or preferably 40x magnification.
  • For fluorescent assay, image acquisition plays an important role in downstream data analysis. Please follow the guidelines and webinar below.


Fluorescent Imaging Recommendations


Filter set recommendations

Selecting the right fluorophore allows proper visualization of the target marker. Please see below for reference:

*These v2 dyes are TSA-conjugated. TSA dyes are available from Akoya Biosciences

For more details on imaging and microscopy, please see this webinar Click Here


Data analysis guidelines

In this next section we provide data analysis guidelines for several types of RNAscope staining results,
including semi-quantitative and quantitative analysis methods for each of the various gene expression

RNAscope and BaseScope assays generate punctate dots each of which represents a single mRNA
molecule. Overlapping mRNA molecules can lead to the signal appearing as clusters as opposed to single
dots. Variations in dot sizes and color intensities can be related to differences in the number of ZZ
probes that are bound to target molecule. Pretreatment optimization may be needed for best signal-to-
noise ratio.

Semi-Quantitative scoring can be performed by visual assessment and counting the dots per cell and bin
them based on number of dots per cell. For more information, please follow the guidelines in the link:

For more Information on Quantifying your results with RNAscope and BaseScope assays using Cell
Profiler Click Here


Additional resources

The RNAscope Assay Data Analysis Webinar
Live demo of using ImageJ to score chromogenic singleplex images
Available at

Get the Most Out of Your RNAscope  Multiplex Fluorescent assays Webinar
Part 2 focus on data interpretation using Image J
Available at

Optimize Your RNAscope Assay Data Analysis Webinar
Deep dive of our data analysis guide
Available at

Become an RNAscope Expert: RNAscope Data Interpretation Webinar
Learn data interpretation with our Chief Medical Officer and board-certified pathologist
Available at

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