Evaluation of DNA Extraction Methods and Real Time PCR Optimization on Formalin-fixed Paraffin-embedded Tissues


Formalin-fixed, paraffin-embedded (FFPE) tissues are the most invaluable source of diagnostic material forstudying pathogenesis of cancer and a variety of other diseases. Unfortunately, DNA extracted from formalinfixed tissues is highly degraded due to cross-linking between nucleic acid strands. Real Time PCR has become thestandard for gene copy as well as RNA transcript determination. Thus, optimum standardization of Real TimePCR is crucial for obtaining accurate quantification for both research as well as for clinical diagnosis. Howeverthere are various factors which have negative impact . The aim of our study was to establish a simpler method ofextraction and Real Time PCR Optimization for FFPE extracted DNA. Five breast cancer tissues that wereformalin fixed and paraffin embedded were used for DNA extraction with four different methods. ExtractedDNA was amplified with different primer sets that gave amplimers of different size. Optimization of Real TimePCR for EMSY, cyclin D1 and β-globin genes was carried out on DNA obtained using heat treatment protocol forannealing temperature, primer concentration and template concentration. Highest quantity of DNA was obtainedwithout the use of expensive reagents and in short time frame. PCR positivity was observed in case of shorteramplimer 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 β-globin genes were 60°C, 60°C and 61°C respectively.Good results were seen with a primer concentration of 300 nM and 5 ng of template DNA. This study indicatesthat DNA obtained from formalin fixed paraffin embedded tissue is highly fragmented and can be used forsuccessful amplification of shorter amplification products up to 250 bp in length. Optimization of real time PCRis important, especially while using SYBR green dye chemistry.