Triplex Sizing Assay
As part of the Vanderbilt-Ingram Cancer Center's Personalized Cancer Medicine Initiative (PCMI) a triplex sizing assay was developed for non-small cell lung cancer (NSCLC) to prioritize anti-cancer therapy (2). This assay can assess insertions and deletions in EGFR exon 19 and exon 20 and HER2 Exon 20 and is based on capillary electrophoresis of fluorescently labeled PCR products (Figure 2).
The ITR is performing this sizing assay on human tumors (formalin-fixed paraffin-embedded (FFPE) and fresh frozen) and cell lines for the research community.
ITR requires only ONE of the following per sample:
20 ng genomic DNA (quantified by a double-stranded DNA detection assay-e.g., PicoGreen® ) or 40 ng genomic DNA (quantified by NanoDrop™)
FFPE tissue (block OR 2-10 um sections from a 1cm x 1cm tumor)
Frozen cell pellet from > 6 cm dish
Figure 2. Triplex Sizing Assay. Multiplex PCR is performed using HEX or FAM labeled fluorescent reverse primers and the resulting PCR products are subject to capillary electrophoresis. Vertically-dashed lines represent the location of the wild-type (WT) peaks for each primer set. Red arrows denote the location of EGFR or HER2 insertions or deletions.