Evaluation of DNA extraction methods and real time PCR optimization on formalin-fixed paraffin-embedded tissues.

ABSTRACT

Formalin-fixed, paraffin-embedded (FFPE) tissues are the most invaluable source of diagnostic material for studying pathogenesis of cancer and a variety of other diseases. Unfortunately, DNA extracted from formalin fixed tissues is highly degraded due to cross-linking between nucleic acid strands. Real Time PCR has become the standard for gene copy as well as RNA transcript determination. Thus, optimum standardization of Real Time PCR is crucial for obtaining accurate quantification for both research as well as for clinical diagnosis. However there are various factors which have negative impact . The aim of our study was to establish a simpler method of extraction and Real Time PCR Optimization for FFPE extracted DNA. Five breast cancer tissues that were formalin fixed and paraffin embedded were used for DNA extraction with four different methods. Extracted DNA was amplified with different primer sets that gave amplimers of different size. Optimization of Real Time PCR for EMSY, cyclin D1 and beta-globin genes was carried out on DNA obtained using heat treatment protocol for annealing temperature, primer concentration and template concentration. Highest quantity of DNA was obtained without the use of expensive reagents and in short time frame. PCR positivity was observed in case of shorter amplimer up to 250 bp in length. Amplimers of higher length failed to amplify with paraffin extracted DNA. Optimum annealing temperature for EMSY, Cyclin D1 and beta-globin genes were 60 degrees C, 60 degrees C and 61 degrees C respectively. Good results were seen with a primer concentration of 300 nM and 5 ng of template DNA. This study indicates that DNA obtained from formalin fixed paraffin embedded tissue is highly fragmented and can be used for successful amplification of shorter amplification products up to 250 bp in length. Optimization of real time PCR is important, especially while using SYBR green dye chemistry.