In situ Hybridization (ISH) Services

XpressWay^TM Profiles and CustomMapping^TM provide a high level assessment of the expression patterns of genes in human tissue. However, it is also important to identify the expression patterns of target mRNA at the cellular level to gain a greater understanding of the potential function of that gene and its possible role in disease.

Asterand has been providing high quality ISH data to our customers for over 10 years. Our extensive human tissue biorepository allows us immediate access to well characterized, high quality human tissues representing a range of therapeutic areas, enabling us to generate ISH data quickly. We have experience in providing ISH services to support oncology, respiratory, inflammation and metabolic disease research.

Stage 1: Assay optimization / validation

Quality Control

• Confirmation of single riboprobe bands by Agilent gel
• DIG and FITC incorporation analyzed by membrane detection
• Membrane hybridization assay at low stringency to confirm hybridization specificity

Optimization

• Frozen tissue sections (FFPE also available)
• Positive control tissue (often selected based on XpressWay^TM Profile data for each target)
• Up to 5 concentrations of proteinase K digestion
• Generation of DIG or FITC labeled riboprobes
• Up to 3 riboprobe concentrations
• 3 hybridization temperatures
• Sense probe controls run alongside each antisense probe
• Assay control, using insulin mRNA hybridization in sections of pancreas

Typical optimization of ISH assay parameters using 3 riboprobe concentrations (sense and antisense), 3 proteinase K concentrations and 3 hybridisation temperatures. Optimal conditions for this target are highlighted.

Stage 2: Assay

• Validated ISH protocol with matching sense probe controls
• Positive control tissue for target mRNA included
• Number of donors and tissue types chosen by the customer
• Flexible reporting options are available:
  • representative region-matched images of antisense probe with sense probe controls
  • description of the expression pattern and intensity of target staining
  • identification of stained structures
  • discussion and conclusions

• Assay slides can be provided for review and analysis

Insulin mRNA expression in human islet b cells, generated using a DIG labelled riboprobe.

Human Tissue Microarrays
The throughput of ISH studies can be vastly increased with the use of tissue microarrays (TMAs). Asterand has expertise in the construction of custom made frozen and FFPE human (TMAs).

To download details of Asterand's TMA approaches for target localization, click here.

Fluorescence in situ Hybridization (FISH)
Asterand has recently established FISH capability in-house for detecting multiple gene copies.

For further information, contact us at advantage@asterand.com