Input Requirements

Input Requirements

Formalin-fixed, paraffin-embedded (FFPE) human tissues are a valuable source of material for molecular analysis and clinical studies. A number of processes and protocols now exist for the extraction and purification of nucleic acids from FFPE samples; however, as the assays used to evaluate DNA and RNA have evolved from simple monoplex PCR to higher plexity products, the quality and amount of nucleic acid extracted from FFPE material has become more critical to the success of these assays.

The TruSight Tumor assay can be used to generate sequencing libraries that are highly multiplexed at both the target and sample level. The high level of assay complexity is enabled by combining an oligo extension/ligation process with universal PCR. Both of these reactions require a largely intact DNA template that can be denatured. The formalin fixation and paraffin embedding of tissues impacts this requirement by fragmenting, cross-linking, and otherwise damaging DNA through a variety of chemical modifications. Assessing the extent of this damage and adjusting the DNA extraction procedure to compensate for it as much as possible is essential to improving the success of the TruSight Tumor Sample Preparation assay with FFPE DNA.

DNA Extraction Recommendations

Illumina recommends Qiagen Supplementary Protocol: Purification of genomic DNA fromFFPE tissue using the QIAamp DNA FFPE Tissue Kit and Deparaffinization Solution for the purposes of extracting the highest amount of amplifiable DNA from an FFPE tissue block, with the following modifications:

  • Extract gDNA from 8 separate 5 μM FFPE tissue sections.
  • Deparaffinize with 320 μl of Qiagen Deparaffinization Solution.
  • Digest with proteinase K in a thermomixer overnight at 1,000 rpm.
  • Decrease elution volume to 30 μl to maximize DNA concentration.