RNAscope®, developed by Advanced Cell Diagnostics, Inc. (Newark, CA, USA), is a novel and increasingly popular technology for the in situ analysis of RNA within fixed tissues.1 Without disturbing tissue morphology, it enables the localisation of specific targets within RNA, examination at the level of the single molecule, and the simultaneous suppression of background noise. By applying RNAscope® to organotypic cell cultures (which use a three-dimensional organisation of cells that more accurately represent the morphological, physiological, and molecular aspects of tissues), it has the potential to provide a window into gene expression as it occurs in the human body. This could have important implications in a number of fields, including molecular pathology, toxicological analysis, drug discovery, and drug development. In addition, since the approach relies exclusively on in vitro techniques and tissues provided by human donors, it supports the internationally-recognised “3Rs” of animal research: “replacement, reduction, and refinement”.2  

Quantifying Optimisation

Philip Morris International presented the results of a study which sought to determine the optimised conditions for RNAscope® and assess its ability to detect and visualise cell-specific RNA in human organotypic nasal epithelial cultures.3 The experimental process was divided into three steps:

  1. Histology: Slides containing five replicates of nasal cultures were stained to assess tissue morphology.
  2. Optimisation: Optimisation focused on: i) pretreatment of cultures, ii) incubation with protease to unmask target RNA, and iii) amplification to increase sensitivity.
  3. Quantification: Slides were scanned to create digital images. Quantification was then carried out with a custom-built solution designed by Definiens, AG. (Munich, Germany), involving the automated detection of relevant tissue areas, nucleus detection, cell simulation, cell and nuclei visualisation, and spot detection.

A scoring system from zero to four, based on the number of spots per cell, was used to evaluate the results of the initial staining. Scores for positive and negative controls determined whether the RNAscope® technique was optimised (a probe detecting RNA of the human Peptidyl-Prolyl Cis-Trans Isomerase B (PPIB) gene was used as a positive control, while a probe for RNA of the bacterial gene 4-hydroxy-tetrahydrodipicolinate reductase (dapB) was used as a negative control). From the initial microscopic evaluation, a strong expression level of the positive control was observed with an absence of background noise, while no expression of the negative control was observed. The average quantification score for the positive control was three, and for the negative control zero, confirming that the RNAscope® technology for human organotypic nasal epithelial cultures was optimised (low values of standard deviation suggested that there was no variability between the five replicates).

Applications

The study shows that RNAscope® can be optimised for the detection and visualisation of cell-specific RNA in human organotypic nasal epithelial cultures, with no disturbance to the RNA or to tissue morphology. This is crucial, as preserving RNA and tissue morphology in vitro ensures an accurate representation of human biology. The technique has the potential to facilitate rapid, accurate, and reliable RNA detection and localisation, and thus has important implications across a variety of fields in molecular biology. At Philip Morris International, for example, we have conducted a range of in vitro organotypic studies looking at the effects of the aerosols of Reduced-Risk Products* on epithelial cells of the bronchus, oral cavity, and nasal passages. RNAscope® may prove to be a valuable complement to the gene expression data generated from these studies.

Laurent Neau, M.Sc., Philip Morris International

 

About Laurent Neau

Laurent Neau is Lead Technician, Tissue Research Laboratory, Philip Morris International. He holds an M.Sc. in Biotechnologies from Lille University, France. He has worked as a technician in histology labs for seven years in both academic research and pharmaceutical company settings. He has developed strong expertise in molecular biology, immunohistochemistry and in situ hybridisation.

Where can readers find more information?

Comprehensive information on PMI’s research and development programs can be found online at www.pmiscience.com.

* Reduced-Risk Products (RRPs) is the term we use to refer to products that present, are likely to present, or have the potential to present less risk of harm to smokers who switch to these products versus continued smoking.  We have a range of RRPs in various stages of development, scientific assessment and commercialisation. Because our RRPs do not burn tobacco, they produce far lower quantities of harmful and potentially harmful compounds than found in cigarette smoke.

References

  1. Wang, F. et al. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. Journal of Molecular Diagnostics 14(1), 22-9. Available online at: https://jmd.amjpathol.org/article/S1525-1578(11)00257-1/fulltext.
  2. National Center for the Replacement, Reduction, and Refinement of Animals in Research. The 3Rs. Available online at: https://www.nc3rs.org.uk/the-3rs.
  3. Neau, L. et al. 2018. Optimisation and image analysis of RNAscope® technology on 3D human organotypic ciliated respiratory epithelial culture. 5th Digital Pathology Congress, Europe. 6-7 December 2018. London, UK.

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