Comparative Assessment of DNA Extraction Techniques From Formalin-Fixed, Paraffin-Embedded Tumor Specimens and Their Impact on Downstream Analysis.

Abstract

Good-quality nucleic acid extraction from formalin-fixed, paraffin-embedded (FFPE) specimens remains a challenge in molecular-oncopathology practice. This study evaluates the efficacy of an in-house developed FFPE extraction buffer compared with other commercially available kits. Eighty FFPE specimens processed in different surgical pathology laboratories formed the study sample. DNA extraction was performed using three commercial kits and the in-house developed FFPE extraction buffer. DNA yield was quantified by a NanoDrop spectrophotometer and Qubit Fluorometer, and its purity was measured by the 260/280-nm ratio. A fragment analyzer system was used for accurate sizing of DNA fragments of FFPE DNA. The downstream effects of all extraction methods were evaluated by polymerase chain reaction (PCR) and Sanger sequencing. In comparison with the commercial kits, the in-house buffer yielded higher DNA quantity and quality number (P < .0001). In addition, DNA integrity and fragment size were preserved in a significantly greater number of samples isolated with the in-house buffer (P < .05). The target PCR amplification rate with the in-house buffer extracted samples was also significantly higher, with 98% of the samples showing interpretable sequencing results. The in-house developed FFPE extraction buffer performed superior to other methods in terms of suitability for downstream applications, time, cost-efficiency, and ease of performance.