5.4. FFPE Guidelines

• We recommend using the Qiagen All Prep DNA/RNA FFPE kit. With FFPE the yield and quality of isolated DNA/RNA can vary significantly, depending on the age and method used to fix the sample.
• Certain impurities and high concentrations of FFPE-DNA can inhibit DNA polymerase activity for AIR-DNA profiling assay. To identify the optimal FFPE-DNA input amount, we suggest testing a range of DNA inputs (e.g., 0.5 µg, 1 µg, 2 µg, 5 µg) for at least one sample (e.g., sample with a high yield of purified DNA), in our experience 1-2 µg can be used for AIR-DNA assay without inhibition of DNA polymerase activity.

Protocol Optimization:

• Considering that FFPE samples usually have very different contents of T/B cells you could expect that only a fraction of samples will generate the expected correct TCR/BCR amplified products. Therefore, we recommended running 1st PCR for 26 cycles and using an aliquot from 1st PCR (2µl) to run 2nd PCR (50 ul) with index primers for 8 cycles. Analyze the yield of PCR products using a Bioanalyzer (or 3% agarose-TAE gel). Collect in the new test tubes 2nd PCR samples that demonstrate visible yield of correct TCR/BCR amplicons and run the rest of the samples (without visible amplified bands) for an additional 4 cycles and repeat Bioanalyzer analysis. Collect samples with amplified correct TCR/BCR amplicons and run an additional 4 cycles for samples without visible band. Based on our experience, the maximum number of PCR cycles in the 2nd PCR step could be 20 cycles. Using more than 20 cycles in the 2nd PCR, in general, is not recommended as 20 cycles could amplify PCR products from only a few template molecules. If you can’t see amplified products after 20 cycles (of 2nd PCR) it could be an indication that FFPE samples practically don’t have T/B cells or DNA is significantly damaged. In those cases, we recommend staining the slides to identify the content of T or B cells. Don’t over-cycle (using more cycles than necessary to amplify visible correct amplicons) the samples in 2nd PCR step. Don’t use more than 2ul of 1st PCR products in the 2nd PCR step (50ul), it could increase the amount of background, non-specific products.