Question: What are tips and best practices for using thermal cyclers with Xenium?
Answer: Tips and best practices for Xenium slide incubation are covered in the following documentation:
- Xenium In Situ for FFPE Tissues – Deparaffinization & Decrosslinking
- Xenium In Situ for Fresh Frozen Tissues - Fixation & Permeabilization
- Assay User Guides
A few important general best practices are covered here:
1) Always use the appropriate Xenium Thermocycler Adaptor associated with each assay chemistry.
! Xenium v1 and Xenium Prime have different thermocycler adaptors. Swaps will result in assay failure.
Xenium v1 (PN-1000623)
Xenium Prime (PN-1000739)
2) Always place two Xenium slides in cassettes on the Xenium thermocycler adaptor.
Xenium v1
Xenium Prime
The deep-well block allows the Xenium Thermocycler Adaptor to sit directly on top of the block for efficient heat transfer to the Xenium slide. Not using a deep-well block will lessen the heat transfer from the block to the slide and result in a decrease in assay performance.
3) Ensure equipment is level.
A small bubble level can ensure the thermocycler adaptor is level for optimal assay performance. Using an instrument that is not level (as shown below) risks improper cassette sealing, evaporation, and/or uneven reagent distribution.
Please refer to thermal cycler manual for leveling instructions.
4) Pay attention to lid temperature.
The lid temperature of some thermal cyclers (Veriti, not officially supported, and XT96, v1 only) do not go lower than 50°C (Veriti) or 40°C (XT96) , but some steps of the Xenium workflow calls for lower lid temperatures.
For those steps that require lower lid temperatures, we would advise turning the lid to lowest temperature possible.
'!' For the XT96, the lid will deactivate below 35°C and lead to condensation during amplification. To mitigate this, go in to System > Thermal Block > General Parameter and manually set the 'Lid Deact' field, highlighted below, to 20°C.
After lid deactivate setting is changed, the lid should be programmed to the temperature specified in the User Guide.
Alternatively, if it is not desirable to modify the XT96 general parameters, the amplification step can be set up as an incubation. It is very important if the thermal cycler is programmed as an incubation, the incubation times of 2:00:00 are followed exactly. Extending incubation times for either ligation or amplification may significantly compromise assay performance.
5) Significant evaporation can compromise assay performance.
6) Ensure thermocycler adaptor and back of slide are free of debris during incubations.
Debris in between the Xenium slide and the Xenium thermocycler adaptor can increase the risk of slide cracking on validated thermal cycler models. The risk of slides cracking with debris is higher during high-temperature steps.
For these steps, we would recommend the following best practices:
- Before placing slides on the thermocycler adaptor, wipe the thermocycler adaptor and the back of the Xenium slide with lens paper wetted with 70% ethanol or isopropanol.
- ‘!’ Avoid kimwipes as they are not lint-free.
- The thermocycler adaptor can also have debris removed with tape, but this is not recommended on the back of Xenium slides.
C1000 Best Practices:
Additional notes:
If using thermal cyclers other than BioRad C1000, ramp rates should be adjusted for all the steps as described below:
- Analytik Jena Biometra TAdvanced: 2°C/sec heating and cooling
Information on which models are compatible and which models are unsupported can be found here: Which thermal cyclers are compatible with Xenium.
Product: Xenium In Situ Gene Expression, Xenium Prime In Situ Gene Expression