DNA Extraction and FFPE

Table of Contents

Letter from the Editor
Index of Experts
Q1: Do you pre-treat your samples prior to extraction?
Q2: What extraction method do you use to isolate DNA from the tissue? How long do you incubate and what buffer do you use?
Q3: How do you optimize the efficiency of DNA extraction? What do you consider a good yield?
Q4: How do you determine the quality of the DNA extracted? What degree of fragmentation is acceptable?
List of Resources

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Letter from the Editor

At the Smithsonian Institute, there are shelves full of jars of biological samples they've collected over the years. At last count, scientists there had more than 83 million samples, many of which have been fixed in formalin.

While easily adapted for histopathology and microscopic examination, these formalin-fixed samples are a bit more reluctant to offer up their nucleic acids for examination. DNA and RNA from FFPE samples are notoriously degraded. But don't underestimate researchers working in this field. Within the last few years, new protocols have emerged that are more efficient at extracting DNA and RNA from these tissues, and previously fixed nucleic acids are being amplified through PCR, analyzed as CGH arrays, or used in whole genome amplification studies.

In this installment of Genome Technology's technical guide series, we take a look at how DNA is extracted from formalin-fixed, paraffin embedded tissues — and it's not an easy endeavor. Here, though, our seasoned experts offer their advice and tips from many rounds of refining their methods.

— Ciara Curtin

Index of Experts

Genome Technology would like to thank the following contributors for taking the time to respond to the questions in this tech guide.

Sunil Badve
Clarian Pathology Laboratory
Indiana University School of Medicine

Carol Barone
Histotechnology Core Lab
Nemours-A.I. Dupont Hospital for Children

Mark Bouzyk
Center for Medical Genomics
Emory University School of Medicine

Susan Long
Pathology Core Facility
Ohio State University Medical College

Jeffrey Mason
Department of Biophysics
Armed Forces Institute of Pathology

Q1: Do you pre-treat your samples prior to extraction?

We do not, except melting the wax at minimal temperature.

— Sunil Badve

We normally collect 5 μm sections into a 2 ml screwcap tube. We usually start with three to six sections. In some cases, we have also used 1 mm tissue cores. Initially, we use xylene for three minutes at 50 degrees Celsius to remove paraffin from the sections, followed by two ethanol washes to remove the xylene.

— Mark Bouzyk

I receive 10 μm unstained sections on non-plus slides, which are deparaffinized in xylene and rinsed with ethanol. The air-dried sections are scraped with a scalpel dipped in digestion buffer and placed in a tube with Qiagen ATL buffer and proteinase K solution.

— Susan Long

For FFPE tissue sections there are two pre-treatment options. In the first method, the tissue (10 to 50 mm sections) is taken through the reverse steps of histologic processing by incubation in xylene and a graded series of ethanol solutions (100 percent, 90 percent, and 70 percent) followed by hydration in PBS buffer. There are variations on this protocol, such as using a different series of ethanol solutions (or just 100 percent ethanol), or hydration of the tissue in 1 M sodium thiocyanate overnight. In the second method, the tissue (10 to 20 mm sections) is heated in buffer at 120 degrees Celsius for 20 minutes and then cooled to room temperature using the method of Shi et al. The buffer contains 28.6 mM each of citric acid, potassium phosphate, boric acid, and diethylbarbituric acid, adjusted to pH 9 with sodium hydroxide. This pre-treatment method also allows you to omit the enzyme digestion step in the subsequent DNA extraction method.

— Jeffrey Mason

Q2: What extraction method do you use to isolate DNA from the tissue? How long do you incubate and what buffer do you use?

We make use of Qiagen DNeasy (or similar product).

— Sunil Badve

We cut the FFPE samples at 10 μm/30 sections into an Eppendorf tube, instead of mincing the tissue. The Molecular Diagnostics Lab adds 300 μl of histo-clear to the tube to remove paraffin (five to 10 minutes/RT) and centrifuges the tube to pellet. Repeat this process three times, discarding the histo-clear/paraffin. Hydrate with 100 percent ethanol/RT. Centrifuge/decanting ethanol three times. Add 300μl lysis buffer, 1.5 μl proteinase K, and incubate for three hours. Repeat or do an over-night incubation, if needed (not usually). MDL uses a protocol from Gentra. The main difference is cutting the 10 μm sections instead of mincing.

— Carol Barone

Once the samples are de-paraffinized, we digest the proteins bound to the DNA. As DNA tends to crosslink quite densely with histones and is generally quite robust (it does not fragment as easily as RNA), we would recommend incubating for a minimum of 48 hours at 50 degrees Celsius with Ambion's Protease and Digestion buffer. We have tried a number of kits and protocols and prefer to use Ambion's RecoverAll Total Nucleic Acid Extraction Kit. For the DNA isolation and purification, we utilize Ambion's proprietary isolation buffers and glass-fiber filters, their washing and elution buffers, and follow their protocol. The whole manual hands-on process is about 45 minutes. For higher throughput, the RecoverAll MagMAX Custom kit is ideally suited for automation purposes.

— Mark Bouzyk

I use the Qiagen QiaAmp DNA Micro Kit for DNA isolation. I incubate the tissue in Qiagen QiaAmp ATL digestion buffer and proteinase K solution at 65 degrees Celsius overnight, at least, and up to 48 hours, adding an additional aliquot of proteinase K and additional hour of incubation prior to extraction.

— Susan Long

The most important step in DNA extraction from FFPE tissues is the protein digestion step. Proteinase K is the most commonly used protease. The digestion/lysis buffer typically contains 10 to 20 mg/mL proteinase K in Tris buffer, pH 8. Generally, a detergent is also added, such as 10 percent SDS. The tissue is heated in the digestion/lysis buffer at about 55 degrees Celsius for 24 hours followed by incubation for five minutes at 95 degrees Celsius to inactivate the protease. Phenol/chloroform/isopropanol (25/24/1) is added to the digestion mixture and the supernatant is recovered by centrifugation. Chloroform is then added to the supernatant and, after centrifugation, the resulting aqueous upper phase is carefully removed. A small volume of 3 M sodium acetate and isopropanol are added and the DNA is precipitated by incubation at -20 degrees Celsius overnight. Glycogen can be added to aid in the precipitation of small amounts of DNA. The precipitated DNA is washed with ethanol, dried, and dissolved in 20 mL to 50 mL of water or buffer. The DNA may require overnight incubation in order to completely dissolve.

— Jeffrey Mason

Q3: How do you optimize the efficiency of DNA extraction? What do you consider a good yield?

Longer incubation with proteolytic enzyme. Greater than 100 ng/μl is considered good yield since it meets the concentration requirement to run GG assay.

— Sunil Badve

The key point in the process is performing the initial protease digestion for between two to three days. We have found that the protocol is not so efficient if protease incubation is a few hours. From our experience, we believe a good yield is typically 1 μg to 3 μg from three 5 μm sections, although this will, to a large extent, depend on the quality and the usable amount of the sections.

— Mark Bouzyk

Make sure the tissues are thoroughly digested prior to running through the QiaAmp column. From 10 to 20 small tissue sections (less than 5 mm2) around 5 μg to 10 μg of DNA and three to five large tissue sections (greater than 5mm2) 40 ug of DNA or greater.

— Susan Long

The extraction efficiency is strongly dependent upon the proteinase K digestion step. Prolonged digestion can improve both the efficiency of DNA recovery and the average length of the DNA fragments. For example, the proteinase K digestion step can be extended for up to five days with fresh digestion buffer added each day. The key is to continue the digestion step until the tissue is completely dissolved. The end product should be a translucent solution with a silky appearance with no visible particulates. The incubation time required for the digestion step is also dependent upon the tissue. For example, a lower-density tissue (lung) will require a shorter incubation time than a high-density tissue (heart).

A good yield for DNA extracted from FFPE tissue is generally 200 to 500 mg/mL. DNA concentration can be measured by absorbance or by fluorescence assay.

— Jeffrey Mason

Q4: How do you determine the quality of the DNA extracted? What degree of fragmentation is acceptable?

1.9 <= A260/A280 <= 1.7. For FFPE DNAs, there is no way to know if the extract is too fragmented or not. We just have to run GG assay to see.

— Sunil Badve

We normally use PicoGreen quantitation to measure the amount and quality of DNA. For most of our downstream assays, which are typically TaqMan or single base extension genotyping, fragmentation down to 500 bp is acceptable. However, there may be cases where you might want longer fragments (e.g. 2 Kb for Illumina Infinium) so we would recommend using Agilent's Bioanalyzer to measure fragment size as it requires very small amounts of starting material (as opposed to routine agarose gel electrophoresis, which typically consumes 1 μg DNA).

— Mark Bouzyk

I check concentration and 260/280 ratio on a Nanodrop spectrophotometer and PCR with a size ladder amplification control. I typically need to be able to amplify 200 bp to 400 bp fragments.

— Susan Long

We use two methods to estimate the quality of DNA extracted from FFPE tissue. The first is to denature the DNA at 70 degrees Celsius and then run about 200 ng on a 1 percent agarose gel to check the size distribution. The size distribution should range from 100 bp to 3,000 bp. Longer fragments may be seen if the proteinase K digestion is extended out to six days. The second method to estimate the quality of the extracted DNA is by real-time PCR using a housekeeping gene, such as GAPDH or ß2- microglobin. We run assays using three to four sets of primers and one to two probes.

For the real-time PCR assays we run, the typical 100 bp to 3,000 bp DNA fragments obtained using our protocol are satisfactory. Longer fragments may be required for DNAarray studies.

— Jeffrey Mason

List of resources

DNA can be extracted from formalin-fixed paraffin-embedded tissues in different ways and for a variety of experiments. Here's a list of resources to help you out when you have to tweak your extraction technique for optimal results.

Publications

Aviel-Ronen S, Qi Zhu C, Coe BP, Liu N, Watson SK, Lam WL, Tsao MS. (2006) Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues. BMC Genomics. 7:312.

Battifora H. (1991) Effect of fixatives and fixation times on tissues. [comment on Greer et al. (1991) Am J Clin Pathol. 95(2):117-24]. Am J Clin Pathol. 96(1):144-5.

Coudry RA, Meireles SI, Stoyanova R, Cooper HS, Carpino A,Wang X, Engstrom PF, Clapper ML. (2007) Successful application of microarray technology to microdissected formalin-fixed, paraffin-embedded tissue. J Mol Diagn. 9(1):70-79.

Coura R, Prolla JC, Meurer L, Ashton-Prolla P. (2005) An alternative protocol for DNA extraction from formalin-fixed and paraffin wax-embedded tissue. J Clin Pathol. 58(8):894-5.

Fox EA. (2001) Preparation of DNA from fixed, paraffin-embedded tissue. Curr Protoc Hum Genet. Appendix 3I.

Gilbert MTP, Haselkorn T, Bunce M, Sanchez JJ, Lucas SB, Jewell LD, Van Marck E, Worobey M. (2007) The isolation of nucleic acids from fixed, paraffin-embedded tissues- Which methods are useful when? PLoS ONE 2(6):e537.

Gillio-Tos A, De Marco L, Fiano V, Garcia-Bragado F, Dikshit R, Boffetta P, Merletti F. (2007) Efficient DNA extraction from 25-year-old paraffin-embedded tissues: study of 365 samples. Pathology. 39(3):345-8.

Greer CE, Peterson SL, Kiviat NB, Manos MM. (1991) PCR amplification from paraffinembedded tissues: effects of fixative and fixation time. Am J Clin Pathol. 95(2):117-24.

Legrand B, Pd M, Durigon M, Khalifat V, Crainic K. (2002) DNA genotyping of unbuffered formalin-fixed, paraffin-embedded tissues. Forensic Science International. 125 (2-3):205B.

Lehmann U, Kreipe H. (2001) Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies. Methods. 25(4):409-18.

Little SE, Vuononvirta R, Reis-Filho JS, Natrajan R, Iravani M, Fenwick K, Mackay A, Ashworth A, Pritchard-Jones K, Jones C. (2006) Array CGH using whole genome amplification of fresh-frozen and formalin-fixed, paraffin-embedded tumor DNA. Genomics. 87:298-306.

McSherry EA, McGoldrick A, Kay EW, Hopkins AM, Gallagher WM, Dervan PA. (2007) Formalin-fixed paraffin-embedded clinical tissues show spurious copy number changes in array-CGH profiles. Clin Genet. 72: 441-447.

Ren Z, Sällström J, Sundström C, Nistér M, Olsson Y. (2000) Recovering DNA and optimizing PCR conditions from microdissected formalin-fixed and paraffin-embedded materials. Pathobiology. 68:215-217.

Shi SR, Cote RJ,Wu L, Liu C, Datar R, Shi Y, Liu D, Lim H, Taylor CR. (2002) DNA extraction from archival formalin-fixed, paraffinembedded tissue sections based on the antigen retrieval principle: heating under the influence of pH. J Histochem Cytochem. 50(8):1005-11.

Shi SR, Datar R, Liu C,Wu L, Zhang Z, Cote RJ, Taylor CR. (2004) DNA extraction from archival formalin-fixed, paraffin-embedded tissues: heat-induced retrieval in alkaline solution. Histochem Cell Biol. 122(3):211-8.

Talaulikar D, Gray JX, Shadbolt B, McNiven M, Dahlstrom JE. (2008) A comparative study of the quality of DNA obtained from fresh frozen and formalin-fixed decalcified paraffin-embedded bone marrow trephine biopsy specimens using two different methods. J Clin Pathol. 61(1):119-23.

Thompson ER, Herbert SC, Forrest SM, Campbell IG. (2005) Whole genome SNP arrays using DNA derived from formalinfixed, paraffin-embedded ovarian tumor tissue. Hum Mutat. 26(4):384-9.

Wu L, Patten N, Yamashiro CT, Chui B. (2002) Extraction and amplification of DNA from formalin-fixed, paraffin-embedded tissues. Appl Immunohistochem Mol MorpholI. 10(3):269-74.

Books

Molecular Diagnostics: For the Clinical Laboratorian. Ed. by William B. Coleman, Gregory J. Tsongalis. (August 2005) Humana Press; ISBN 9781588293565.

Molecular Pathology Protocols (Methods in Molecular Medicine). Ed. by Anthony A. Killeen. (January 2001) Humana Press; ISBN: 9780896036819.

Morphology Methods: Cell and Molecular Biology Techniques. Ed. by Ricardo V. Lloyd. (June 2001) Humana Press; 9780896039551.

Path to Effective Recovering of DNA from Formalin-Fixed Biological Samples in Natural History Collections: Workshop Summary. By Evonne Tang. (January 2006) National Academies Press; ISBN: 9780309102933.

Conferences

NCRI Cancer Conference
October 5-8, 2008; Birmingham, UK

Discovery 2 Diagnostics
October 20-22, 2008; San Diego, CA

AACR Annual Meeting
April 18-22, 2009; Denver, CO