Lineage-specific Next Gen Multiplex In Situ Hybridization for HIV or SIV RNA and DNA

• Performs HIV and SIV in situ hybridization to detect vRNA and vDNA. 
• Uses specialized technologies and probes to analyze tissue samples. 
• Aims to provide a better understanding of the predominate location of viral reservoirs within tissue microenvironments.
• Provides relevant data electronically alongside a report with interpretations and conclusions.  
• Includes iterations depending on your needs: 

The process

• The Tissue Analysis Core will process, embed, and perform microtomy on fixed tissue samples presented in ethanol. 
• HIV/SIV in situ hybridization for detection of vRNA and vDNA will be performed using the next-generation RNAscope technology platform using lineage specific in situ hybridization probes and kits designed and generated by Advanced Cell Diagnostic, Inc. 
  • These in situ hybridization probes and kits have been designed to increase both sensitivity and specificity for the viral lineage of interest. 
• RNAscope and DNAscope will be perform simultaneously on the same tissue section, and then immunofluorescence will be performed to target cellular marker(s). 
  • This multiplex approach that combines RNAscope and DNAscope to identify and localize vDNA+ cells expressing, or not expressing, vRNA in the same section of tissue coupled with the ability to discriminate cell populations based on protein expression could provide a better understanding of the predominate location and phenotype of viral reservoirs within tissue microenvironments. 
• A high-resolution digital image of each sample will be generated using the Keyence BZ-X700 fluorescence digital microscope system or Fluoview FV10i confocal laser scanning system. 
• Under the guidance of a subject matter expert, the core will provide comprehensive representative pictures and quantitative image analysis using Cell Profiler and Keyence algorithm to determine the number and type of vRNA+ and or vDNA+ cells in each high-resolution image. 
  • The number of nuclei for each region of interest will be determined by segmentation using Cell Profiler to be able to report quantification by millions of cells. 

Includes sufficient basic consultation with a subject matter expert.