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Molecular Pathology Protocols

In routine histopathology, most tissues are fixed in formalin and embedded in paraffin for long-term preservation. DNA can be extracted from these tissues for subsequent molecular analysis by amplification methods. We describe herein a protocol for DNA preparation from paraffin-embedded tissues based on published procedures (1–3). In brief, tissue sections are placed into microfuge tubes, then deparaffinized with xylene. The xylene is removed with ethanol washes, and the tissue is treated with proteinase K to make DNA available for amplification | M E T H O D S I N M O L E C U L A R M E D I C I N ETM Molecular Pathology Protocols . M Edited by Anthony A. Killeen 1_ DNA Extraction from Paraffin-Embedded Tissues Hongxin Fan and Margaret L. Gulley 1. Introduction In routine histopathology most tissues are fixed in formalin and embedded in paraffin for long-term preservation. DNA can be extracted from these tissues for subsequent molecular analysis by amplification methods. We describe herein a protocol for DNA preparation from paraffin-embedded tissues based on published procedures 1-3 . In brief tissue sections are placed into microfuge tubes then deparaffinized with xylene. The xylene is removed with ethanol washes and the tissue is treated with proteinase K to make DNA available for amplification. This protocol is simple but there are several factors that influence the success of subsequent DNA amplification assays including the type of fixative that is used the duration of fixation the age of the paraffin block and the length of the DNA segment to be amplified see Note 1 . Ethanol fixation preserves DNA much better than does formalin. Formalin fixation randomly chops DNA in a duration-dependent manner resulting in partial degradation. Even more severe DNA degradation occurs in bone samples subjected to acid decalcification. Because of this degradation formalin-fixed tissue is not suitable for Southern blot analysis or for amplification of large DNA segments. Nevertheless polymerase chain reaction PCR amplification of segments ranging up to 1300 bp has been reported 2 and consistent amplification of segments up to 300 bp is commonly achieved from archival fixed tissues. Be aware that partial degradation of DNA may result in sampling bias and therefore results should be interpreted with caution. For example amplification of DNA from one cell may produce a PCR product that is not representative of the entire population of cells in the tissue. For .