After After
Before Before

10099 - RNAscope, Skin Cancer

INTRODUCTION
Presented in 2012, the RNAscope is one of the more recent RNA in situ hybridization techniques that allows visualization of multiple target genes. RNAscope uses a probe design strategy that amplifies the signal and suppresses the background, which results in increased sensitivity and specificity [see REFERENCES: 1].

The protocol automatically detects and quantifies probes associated with a cell nucleus in dermis and epidermis, respectively. No manual outlining of dermis and epidermis is needed, since these regions are automatically identified. 

KEYWORDS
RNAscope, dermis, epidermis, skin cancer, digital pathology, image analysis.

METHODS
The first image processing step involves an automated detection of the dermis and epidermis, i.e. the regions of interest (ROI) [See Example: Figure 2]. The nuclei and probes are subsequently detected inside these ROIs [See Example: Figure 3, Figure 5, Figure 7]. A polynomial blob filter that enhances the probes’ features is used for the identification of the probes, whereas the haematoxylin color-deconvolution band is used for the identification of nuclei. The initial classification of nuclei and probes is managed with a postprocessing protocol to firstly refine the detection of nuclei and probes, and secondly to qualitatively determine if probes are covered by nuclei or not for the final probe quantification. For each tissue type, i.e. dermis and epidermis, probes are counted and categorized as either covered by nuclei or as separate from nuclei.

QUANTITATIVE OUTPUT VARIABLES
The output variables obtained from this protocol include:

- Probes w Nuclei (Epidermis): The number of probes inside nuclei in the epidermis

- Probes wo Nuclei (Epidermis): The number of probes outside nuclei in the epidermis

- Probes w Nuclei (Dermis): The number of probes inside nuclei in the dermis

- Probes wo Nuclei (Dermis): The number of probes outside nuclei in the dermis

AUXILIARY APPS (included)
ROI detection:
APP: “01 Detect ROI”
The auxiliary APP '01 Detect ROI' is used to automatically outline and separate the dermis and epidermis regions of interests for the subsequent analysis.

WORKFLOW
Step 1: Load the auxiliary APP for ROI identification: “01 Detect ROI”.

Step 2: Load the APP for identification and scoring of probes and nuclei in dermis and epidermis: “02 Analyze”.

STAINING PROTOCOL
There is no staining protocol available.

ADDITIONAL INFORMATION
The APP utilizes the Visiopharm EngineTM and Viewer software modules, where EngineTM offers an execution platform to expand processing capability and speed of image analysis. The Visiopharm Viewer allows a fast review together with several types of image adjustment properties ex. outlining of regions, annotations and direct measures of distance, curve length, radius, etc. 
By adding the AuthorTM module the APP can be customized to fit other purposes. AuthorTM offers a comprehensive and dedicated set of tools for creating new fit-for-purpose analysis APPs, and no programming experience is required.

REFERENCES

LITERATURE

1. RNAscope – A Novel in Situ RNA Analysis Platform for Formalin-Fixed Paraffin-Embedded Tissues, F. Wang, J. Flanagan, N. Su, L.C. Wang, S. Bui, A. Nielsen, X. Wu, H.T. V, X.J. Ma and Y. Luo, Journal of Molecular Diagnosis (2012), Jan; 14(1): 22-29. Doi: 10.1016/j.jmoldx.2011.08.002

 

RUO
Figure 1
Figure 1
Raw image. Dermis is predominant with epidermis only as a crust of the dermis in the lower part of the image.
Figure 2
Figure 2
Result of analysis with the Auxiliary ROI APP: "01 Detect ROI". Dermis and epidermis are clearly separated and outlined by a green and a blue ROI, respectively.
Figure 3
Figure 3
Results of analysis with the APP: "02 Analyze". Nuclei inside the dermis and epidermis regions are labeled blue, whereas probes inside the nuclei are labeled green and probes outside the nuclei red.
Figure 4
Figure 4
Raw image zoomed in on group of nuclei.
Figure 5
Figure 5
Result of analysis with the APP: "02 Analyze" on the image from Figure 4. Nuclei are labeled blue, probes inside the nuclei are labeled green and probes outside the nuclei red.
Figure 6
Figure 6
Zoomed in version of figure 4.
Figure 7
Figure 7
Zoomed in version of figure 5. Nuclei are labeled blue, probes inside the nuclei are labeled green and outside the nuclei red.