Fig. 3: Tiered multiplexed ddPCR improves detection of targeted translocations.

A Multiplex digital PCR of FFPE samples from patients with translocation positive sarcoma. Left is the known translocation partner with genes color-coded as in Fig. 1C. The middle of the panel shows the size distribution of the extracted RNA by capillary gel electrophoresis with a vertical dotted line indicating 200 base pairs as a reference and the RNA integrity numbers (RIN) indicated to the right of each histogram⁷. On the right of this panel is shown the ddPCR fluorescent measurements for samples after multiplexed amplification with primer-probe sets for PCR1 and PCR2 reactions. B Schema depicting the addition of pre-amplification using the same primers as used for ddPCR (but without probes) to first amplify cDNA made from targeted fusion partners prior to analysis with ddPCR. C ddPCR results for cDNA made from RNA extracted from a negative control sample (fusion negative cell line, 293T) and the A673 cell line expressing an EWSR1-FLI1 fusion transcript. RNA was serially diluted, prior to making cDNA, to the indicated targeted amount. ddPCR was performed without (left) and with (right) pre-amplification. D Pre-amplification was then applied to the same FFPE samples as in panel A, demonstrating increased ddPCR fluorescence signaling matching the expected pattern for each known fusion type in 8 of 11 samples tested. The three samples that remained negative with pre-amplification are depicted in Fig. 4C.E Examples of translocations detected in samples for which cDNA was made from 1/20^th of the original extracted RNA. F Tiered multiplexed ddPCR performed on A673 and RH4 cells flow sorted into wells with the indicated number of cells. The expected transcript was detected at a single cell level.