Different DNA Extraction Methods for Paraffin-Embedded Pathological Samples

Background and Objectives: Paraffin-embedded tissue specimens are excellent resources for large-scale molecular epidemiological studies, but the extraction of the high quality nucleic acidmay be problematic. The aim of study was identification of the best method for DNA extraction of paraffin-embedded pathological samples. Materials and Methods: In order to identify the optimal method for DNA extraction, DNA extraction was optimized by comparing four different tissue digestion buffers and six differentprotocols (xylene/ethanol method 1, xylene/ethanol method 2, xylene/ethanol method 3, simpleboiling method, microwave method, and thermal cycler heating method) for paraffin elimination. To evaluate the quality and stability of the extracted DNA, they were used to amplify a 260bpfragment from the β-globin gene in PCR methods. Results: Amplification of a 260 bp β-globin gene fragment using tissue digestion protocol 4 wasobtained in 22/25 (88%) of samples in xylene/ethanol method 1, 19/25 (76%) in xylene/ethanolmethod 2, 19/25 (76%) in xylene/ethanol method 3, 20/25 (80%) in simple boiling method, 19/25 (76%) in microwave method, and 0/25 (0%) in thermal cycler heating method. PCR amplification was also done using two-fold serial dilutions of the 10 positive DNA samples and tissue digestionprotocol 4. In this part, different deparaffinisation methods (xylene/ethanol different methods, simple boiling, and microwave methods) were compared. Successful amplification of a 260 bp β- globin gene fragment of 1:10 and 1:20 dilutions was obtained for xylene/ethanol (method 1) andmicrowave method. Multivariate logistic regression statistical test and SAS software were used for statistical analysis. Conclusion: Based on the results, tissue digestion protocol 4, xylene/ethanol (method 1) and microwave protocols are the best.

کلیدواژه ها:

DNA ، Beta-Globin ، Polymerase Chain Reaction ، Paraffin-Embedding