DNA Qualitative and Quantitative Comparison of Alcohol Fixed Cytology Versus Formalin Fixed Tissue for PNA Clamp Real-Time PCR Detection of KRAS, EGFR and BRAF
Frida Rosenblum, Keith Tomaszewicz, Maryann St. Cyr, Andrew Fischer, Ediz F Cosar, Elizabeth M Kurian, Lloyd Hutchinson. University of Massachusetts Medical Healthcare System, Worcester, MA
Background: Formalin-fixed paraffin embedded tissue (FFPE) is the FDA-approved substrate for polymerase chain reaction (PCR) detection of mutations in BRAF and KRAS, and is commonly used for EGFR testing. We evaluate the quality of the desired DNA required for adequate molecular analysis in alcohol fixed cytology specimens, compared to FFPE.
Design: At UMASS, 20 surgical FFPE (8/2009-7/2012) and 20 alcohol fixed cytology cell blocks (1/2011–6/2012) from multiple organ sites were analyzed. Tumor was macrodissected from slides. DNA was extracted (∼10ng) and tested by spectrophotometry, multiplex endpoint QC-DNA assay and PNA clamp real-time PCR for BRAF, KRAS and EGFR (see Table 1). Peripheral blood DNA was used as the quality gold standard. The delta CT was calculated by subtracting the patient DNA CT value from the peripheral blood DNA.
Results: We evaluated the following parameters: DNA yield, OD 260/280 ratio and maximum amplicon size. The range and average values are shown:
|Average DNA yield (range)||1.48 (0.37-4.35)||1.48 (0.10-4.275)|
|Average OD260/280 (range)||1.92 (0.94-2.37)||1.98 (1.66-2.46)|
|Average amplicon size (range)||448 (QNS/100-500)||444 (QNS/300-500)|
|Average Delta CT||-1.5238||-2.82812|