FFPE Tissue Sample Input Recommendations
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To obtain reliable, reproducible results from formalin-fixed paraffin-embedded (FFPE) DNA samples, accurately assess DNA quality to determine the required number of PCR cycles in this protocol. For more information, refer to DNA Input Recommendations.
Use FFPE DNA quality criteria to determine the appropriate input for successful library preparation. For FFPE samples with ∆Cq value of ≤ 5, the recommended DNA input is 50–1000 ng. IDPE is not recommended for poor quality FFPE samples with ∆Cq > 5. Using samples with ∆Cq > 5 is possible, but it might increase chances of library preparation failure or decrease assay performance.
FFPE Extraction
Use a nucleic acid isolation method that produces high recovery yields, minimizes sample consumption, and preserves sample integrity. The QIAGEN AllPrep DNA/RNA FFPE Kit provides high yield of nucleic acids compared to other extraction methods tested for this assay.
FFPE DNA Qualification
For optimal performance, assess DNA sample quality using the Infinium FFPE QC Kit with KAPA qPCR Master Mix (Universal) and Primer Premix. Perform this assessment on the Bio-Rad CFX96 Touch Real-Time PCR Detection System or equivalent instrument. For more information on FFPE DNA qualification, refer to the Infinium HD FFPE QC Assay Protocol (document # 15020981).
FFPE Reference Samples (Optional)
Use characterized reference materials such as Horizon HD799 (DNA) as a positive control when performing the protocol. You can also use qualified FFPE materials from cell line derived xenografts as reference samples. Use a fluorometric-based method to quantify reference materials before use.
Running a positive control reference sample or no template control consumes reagents and reduces the total number of unknown samples that can be processed.
