The TruSight Tumor 170 assay is optimized for a defined RNA/DNA input range. The optimal range for DNA is from 40 ng to 120 ng total or 3.3 ng/µl to 10 ng/µl. The optimal range for RNA is from 40 ng to 85 ng total or 4.7 ng/µl to 10 ng/µl. Quantify the input RNA/DNA before beginning the protocol. To obtain sufficient nucleic acid material, Illumina recommends isolating nucleic acid from a minimum of 2 mm3 of FFPE tissue.
- 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 a high yield of nucleic acids compared to other extraction methods tested for this assay.
- Use a fluorometric quantification method that uses RNA/DNA binding dyes such as QuantiFluor® (RNA) or AccuClear™ (DNA).
- Dilute starting material in RNase/DNase-free water.
For optimal performance, assess DNA and RNA sample quality before using the TruSight Tumor 170 assay.
- DNA samples can be assessed using the Illumina FFPE QC Kit.
- Use DNA samples that result in a delta Cq value ≤ 5. Using samples with a delta Cq > 5 may decrease assay performance.
- RNA samples can be assessed using Advanced Analytical Technologies Fragment Analyzer™ (Standard Sensitivity RNA Analysis Kit) or Agilent Technologies 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit).
- Use RNA samples that result in a DV200 value of ≥ 20%. Using samples with a DV200 value < 20% may decrease assay performance.