We recommend to use control probes to validate the assay workflow, optimize the permeabilisation conditions for your specific tissue samples and validate the RNA integrity in your samples. In order to quickly be successful, we recommend running the assay as follows:
Validate the assay workflow
Test your tissue samples with Malat1 probe (high expressor) in order to validate the assay workflow in your laboratory. For this step, you will use the standard assay conditions known for your samples.
Optimize the permeabilization conditions
Test your tissue samples with Rplp0 probe (low-medium expressor). Only the protease treatment or the target retrieval steps might need optimisation. All other assay steps are standardized.
Use your gene panel on your tissue sample with the optimised assay conditions.
Generate your HS Library with the predefined and/or custom gene panel for ISS.
Depending on the tissue samples and the sample preparation methods, some optimization might be needed for optimal results. CARTANA HS Library Preparation assay conditions are standardized and only the permeabilization step may require optimization. The assay conditions below are suggestions based on in-house and customer experience. Some other conditions or buffer may be suggested depending on tissue samples and sample preparation methods.
It is recommended to work in an RNase free environment to avoid degradation of RNA molecules. Always use fresh RNAse free reagents for samples preparation.