After After
Before Before

10127 - RNAscope, Rat Heart

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[1].

The protocol automatically detects and quantifies probes for a wide range of genes: ANGPT2, EGLN3, GLP1R, HIF1A, PECAM1, SLC2A1 and dapB. Each cell is classified as 0, 1+, 2+, 3+, or 4+ based on the number of probes it contains as recommended by ACD.

KEYWORDS
RNAscope, heart, rat, composite score, digital pathology, image analysis, RNA, ISH, probe, ANGPT2, EGLN3, GLP1R, HIF1A, PECAM1, SLC2A1, dapB

METHODS
The protocol works within a manually outlined region of interest (ROI). Initially, probes and nuclei are detected. Probes are identified using a polynomial blob filter and the haematoxylin color-deconvolution band is used for nuclei identification. The initial classification of nuclei and probes is managed with a postprocessing protocol to refine the detection of nuclei and probes and to simulate cytoplasm. Cytoplasm is simulated by extending nuclei a predefined diameter, or until another cell is encountered. Cells are classified as 0, 1+, 2+, 3+, or 4+ based on the number of probes covered.

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

  • Probes (#): Number of probes
  • Probes area (µm2): Area of probes
  • Tissue area (µm2): Area of outlined tissue
  • Cells 0 (#): Number of cells with 0 probes
  • Cells 1+ (#): Number of cells with 1-3 probes
  • Cells 2+ (#): Number of cells with 4-9 probes
  • Cells 3+ (#): Number of cells with 10-14 probes
  • Cells 4+ (#): Number of cells with more than 15 probes
  • Composite Score: 1x (% 1+ cells) + 2x (% 2+ cells) + 3x (% 3+ cells) + 4x (% 4+ cells).

AUXILIARY APPs
No auxiliary APPs are available.

WORKFLOW
Step 1: Manually outline ROIs.
Step 2: Load and run the APP for quantification of probes and cells.

STAINING PROTOCOL
There is no staining protocol available.

ADDITIONAL INFORMATION
The APP utilizes the Visiopharm Engine™ and Viewer software modules, where Engine™ offers an execution platform to expand processing capability and speed of image analysis. The 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 Author™ module the APP can be customized to fit other purposes. Author™ offers a comprehensive and dedicated set of tools for creating new fit-for-purpose analysis APPs, and no programming experience is required.

REFERENCES

USERS
The APP was developed for Charles Pyke, Camilla Ingvorsen, and Jonas Ahnfelt-Rønne, Novo Nordisk A/S.

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
Field-of-view from the left ventricle of a rat heart stained for EGLN3.
Figure 2
Figure 2
Identified nuclei (blue) and simulation of cytoplasm (yellow).
Figure 3
Figure 3
Classification of cells based on the number of probes – 0, 1+ and 2+ shown (bright, mid and strong blue).
Figure 4
Figure 4
Field-of-view from the left ventricle of a rat heart stained for PECAM1.
Figure 5
Figure 5
Classification of cells based on the number of probes – 0, 1+, 2+ and 3+ shown (bright, mid, strong, and dark blue).