Different DNA Extraction Methods for Paraffin-Embedded Pathological Samples

Paraffin-embedded tissue specimens are excellent resources for large-scale molecular epidemiological studies, but the extraction of the high quality nucleic acid may be problematic. The aim of study was identification of the best method for DNA extraction of paraffin-embedded pathological samples.

In order to identify the optimal method for DNA extraction, DNA extraction was optimized by comparing four different tissue digestion buffers and six different protocols (xylene/ethanol method 1, xylene/ethanol method 2, xylene/ethanol method 3, simple boiling 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 260bp fragment from the β-globin gene in PCR methods.

Amplification of a 260 bp β-globin gene fragment using tissue digestion protocol 4 was obtained in 22/25 (88%) of samples in xylene/ethanol method 1, 19/25 (76%) in xylene/ethanol method 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 digestion protocol 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) and microwave method. Multivariate logistic regression statistical test and SAS software were used for statistical analysis.

Based on the results, tissue digestion protocol 4, xylene/ethanol (method 1) and microwave protocols are the best.