5.4. FFPE Guidelines

• We recommend using the Thermo Fisher’s RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE (Cat.# AM1975). With FFPE, the yield and quality of isolated RNA can vary significantly, depending on the age and method used to fix the sample. It is critical to assess the quality of the RNA before proceeding with the DriverMap protocol.
• We recommend using the maximum amount of input RNA (1-2 ug) for the library prep.
• After extraction, please measure the RIN score with a Bioanalyzer. For successful AIR profiling, we recommend a RIN Score > 5, or FFPE-RNA containing at least 20% of fragments above 300 nucleotides. In samples, where this is not possible, we recommend running an AIR DNA profiling assay as DNA is more stable than RNA. Please refer to the Technology Guide for more information.
• We recommend running TCR and BCR profiling in separate reactions (rather than in one reaction) considering that the content of T and B cells could be very different between different FFPE samples.

Protocol Optimization:

• Considering that FFPE samples usually have very different contents of T/B cells you could expect that only a subset of samples will generate the expected correct TCR/BCR amplified products. Therefore, we recommend running 1st PCR for 24 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 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), as it could increase the amount of background, non-specific products.