1Department of Integrative Oncology, BC Cancer Research Centre, 2Interdisciplinary Oncology Program, University of British Columbia - UBC, 3Photography/Video Production, Multi-Media Services, BC Cancer Agency, 4Department of Pathology and Laboratory Medicine, University of British Columbia - UBC
Pikor, L. A., Enfield, K. S. S., Cameron, H., Lam, W. L. DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses. J. Vis. Exp. (49), e2763, doi:10.3791/2763 (2011).
Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20°C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.
1. Procedural notes
2. Paraffin removal
3. Ethanol rehydration
4. Tissue digestion
5. DNA clean up
*To perform back extractions add 50-100 μl of dH20 to the sample tube containing the interphase and organic portion. Invert the tube to mix, and spin the sample at 14,000 rpm in a microcentrifuge for 5 minutes. Collect the aqueous phase and add it to the previously acquired aqueous extraction. Continue back extractions until the interphase is clear.
6. DNA precipitation
7. DNA quantification
Biopsied or surgically excised tissues for histopathologic analysis and diagnosis are often formalin fixed and paraffin embedded (FFPE) for long term storage. With the growing interest in understanding the genetic basis of disease, the ability to extract DNA from these samples represents an invaluable source of diagnostic material that can be used for genomic analysis and translational studies. Historically FFPE samples were not considered a viable source for molecular analysis as nucleic acids may be heavily modified by protein-nucleic acid and protein-protein cross linking6. However, the discovery that protease digestion releases fragmented nucleic acids which are suitable for downstream analyses including PCR, array CGH, sequencing and methylation profiling, enables the use of these valuable specimens for genetic analysis 6.
DNA extraction from paraffin-embedded tissues is a robust procedure that relies on differential solubility to purify DNA. Extracted DNA quality and quantity and the success of subsequent DNA amplification is dependent on a number of parameters before, during and after extraction. These include, but are not limited to: type and amount of tissue, the type of fixative used for tissue preservation, the duration of fixation, age of the paraffin block and storage conditions, as well as the length of the desired DNA segment to be amplified 1,7. Removal of paraffin from the tissue is the most critical step for successful extraction as undissolved paraffin leads to poor sample quality and inhibition of PCR amplification.
No conflicts of interest declared.
We would like to thank members of the Lam lab for their evaluation and critiques of this video and article. This work was supported by funds from the Canadian Institutes for Health Research.
|Xylene||VWR international||CABDH6216- 4||-|
|1.7 ml SafeSeal Microcentrifuge Tubes||Sorenson BioScience||11510||-|
|Spectrafuge 16M Microcentrifuge||Labnet International||C0160-B||-|
|Lysis Buffer||10 mM Tris-HCl pH8,
100 mM EDTA pH 8,
50 mM NaCl,
200 μg/ml Proteinase K
|Proteinase K Stock Solution||Invitrogen||AM2548||20 mg/ml|
|Proteinase K Stock Solution|
|Buffer Saturated Phenol pH 6.6/7.9||Fisher Scientific||BP1750I-400||-|
|Phenol: chloroform: Isoamylalcohol (25:24:1, pH 6.7/8.0)||Fisher Scientific||BP1752I-400||-|
|RNase A||Roche Group||10109169001||100mg|
|3M sodium Acetate pH 5.2||40.81g NaOAc in 80ml
Adjust to pH 5.2 with glacial acetic acid
Add dH2O to 100ml
|ND 3300 Spectrophotometer||NanoDrop||-||-|