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FAQs
Browse our frequently asked technical questions by selecting a product below.
How do I prepare FFPE samples for extraction with PCRBIO Rapid Extract PCR and Lysis Kits?
FFPE samples should be prepared as follows:
Deparaffinisation:
Remove the paraffin wax from the FFPE tissue section using a solvent like xylene or a specialized buffer.
Xylene is the most widely used solvent in the deparaffinisation of tissue sections. It dissolves the paraffin wax, enabling it to be removed from the tissue. For effective deparaffinization, the slides are typically immersed in xylene or a xylene substitution for 2–3 cycles, with each immersion lasting between 5 and 10 minutes. Xylene is highly effective but also toxic, so appropriate safety measures – such as working in a well-ventilated space or using xylene substitutes – should be followed.
The first immersion into the xylene bath begins dissolving the paraffin. After 5-10 minutes, the slides are transferred to the next bath. A second xylene bath ensures any remaining paraffin is fully dissolved, further improving the quality of deparaffinisation. Researchers may choose to perform a third xylene bath for thicker or older sections.
Rehydration:
After the paraffin is removed, the tissue needs to be rehydrated to restore its original state. This is done by gradually decreasing the concentration of ethanol, which is critical to prevent tissue damage. This step also helps eliminate any residual xylene that may still be present in the sample.
The rehydration process typically follows these 4 steps:
- 100% Ethanol: The slides are immersed in absolute ethanol for 5 minutes to remove any remaining xylene.
- 95% Ethanol: The slides are transferred to a solution of 95% ethanol for another 5 minutes. This step starts the gradual rehydration process.
- 70% Ethanol: The next step involves placing the slides in 70% ethanol for 5 minutes, which further hydrates the tissue.
- 50% Ethanol: The slides are then moved to 50% ethanol for 5 minutes to ensure full hydration.
By the end of this step, the tissue has been completely rehydrated and is ready for further analysis.
Extraction:
Follow our protocol for PCRBIO Rapid Extract products. Typical incubation times could be increased based on the size/type of tissue, up to 48 hours (try few time points to identify optimal conditions when doing this for the first time). Optimal conditions depend on the tissue type and specific workflow.
