The suitability of DNA extracted from formalin-fixed, paraffin-embedded tissues for double differential polymerase chain reaction analysis

Formalin-fixed, paraffin-embedded (FFPE) tissues are one of the popular sou rces of diagnostic materials, the easiest to store and transport. They are often used as the source of nucleic acids for retrospective molecular analy ses based on DNA amplification by polymerase chain reaction (PCR). However, it is known that nucleic acids from paraffin-embedded tissues are much wor se templates than those recovered from fresh tissues. It is exceptionally i mportant in a quantitative analysis, including double differential PCR (ddP CR). Therefore, a pilot study comparing quantity and quality of DNA extract ed with various paraffin removal and DNA isolation procedures from FFPE tis sues was conducted. Furthermore, the suitability of DNA isolated with optim ized procedure for the assessment of erbB-2 average gene copy number (AGCN) was checked. Specimens for comparison of extraction and isolation procedur es were generated from the same human normal thyroid tissue embedded in par affin to eliminate variabilities in tissue processing and sample size. Thre e procedures of paraffin removal and three procedures of DNA extraction fro m deparaffinized tissue were compared. Only one procedure provided DNA, whi ch was efficiently amplified in ddPCR. The material obtained with this opti mized procedure was used to check the precision of ddPCR by evaluation of A GCN of erbB-2 oncogene. Low variability of obtained results close to expect ed AGCN value (AGCN=1) indicates high reproducibility of the method, as wel l as its high accuracy, if the normal value of erbB-2 AGCN in the examined tissue is assumed.