Comparison of methods in the recovery of nucleic acids from archival formalin-fixed paraffin-embedded autopsy tissues

doi:10.1016/j.ab.2010.01.014

Analytical Biochemistry

Volume 400, Issue 1, 1 May 2010, Pages 110-117

https://doi.org/10.1016/j.ab.2010.01.014 Get rights and content

Abstract

Archival formalin-fixed paraffin-embedded (FFPE) human tissue collections are typically in poor states of storage across the developing world. With advances in biomolecular techniques, these extraordinary and virtually untapped resources have become an essential part of retrospective epidemiological studies. To successfully use such tissues in genomic studies, scientists require high nucleic acid yields and purity. In spite of the increasing number of FFPE tissue kits available, few studies have analyzed their applicability in recovering high-quality nucleic acids from archived human autopsy samples. Here we provide a study involving 10 major extraction methods used to isolate total nucleic acid from FFPE tissues ranging in age from 3 to 13 years. Although all 10 methods recovered quantifiable amounts of DNA, only 6 recovered quantifiable RNA, varying considerably and generally yielding lower DNA concentrations. Overall, we show quantitatively that TrimGen’s WaxFree method and our in-house phenol–chloroform extraction method recovered the highest yields of amplifiable DNA, with considerable polymerase chain reaction (PCR) inhibition, whereas Ambion’s RecoverAll method recovered the most amplifiable RNA.

Section snippets

Sampling and laboratory work authentication

We randomly selected seven FFPE autopsy blocks, all visceral tissues involving random organs to mimic archival pathological tissues that would typically be found in most repositories in the developing world. All tissues, according to documentation, were fixed in 10% buffered formalin before paraffin embedment, with embedment dates ranging from 1995 to 2005. The tissues were obtained from the tissue repository at the Department of Pathology, School of Biomedical Sciences, College of Health

Total DNA recovery and c-Myc DNA quantitation

All samples and extraction methods recovered measurable amounts of DNA as assessed by the PicoGreen assay; however, the amounts varied considerably, ranging from 0.02 (0–0.04) ng/μl in Ambion’s TRR method to 5.47 (2.03–7.73) ng/μl in the in-house PCE method (Table 2). Of the 10 methods tested, 6 yielded significantly higher amounts of DNA: (from highest to lowest) the in-house PCE, TrimGen’s WXF, Ambion’s RAD, Stratagene’s ABS, Ambion’s RAR, and Sigma’s GEN (Table 2 and Figs. 1A and 1B). All

Total DNA, amplifiable nuclear DNA copies, and inhibition

Not all 10 methods tested here were originally designed for the extraction of DNA. Despite this, all methods except Ambion’s TRR recovered DNA as measured by the TBS-380 PicoGreen assay. Of these 9 remaining methods, 6 had between 2- and 32-fold more DNA than the other 3 methods. It is important to note that despite detectable DNA within the extracts, this itself was not a guarantee of the successful amplification of single-copy nuclear DNA. In fact, only 4 of the 6 methods yielded more than

Conclusion

The recovery and amplification of nucleic acids from archived formalin-fixed autopsy human tissues is a growing field in retrospective genetic studies. Scientists are faced with the problem of choosing methods that not only are able to recover high amounts of nucleic acids but also yield amplifiable copies. In this study, we have provided a careful test comparison of 10 major FFPE extraction methods on seven randomly collected archival human pathological tissues. Whereas we found that TrimGen’s

Acknowledgments

We thank Kirsten Bos, Linda Rodriquez, and Christine King for their helpful discussions and suggestions on the paper. We also thank E. Othieno, a consultant pathologist at Mulago Hospital in Uganda, for help in diagnostic sorting of the tissues. We are grateful to Ambion (Austin, TX, USA), Roche (Basel, Switzerland), Sigma (St. Louis, MO, USA), and TrimGen (Sparks, MD, USA) for supplying us with their respective FFPE nucleic acid extraction kits or protocols tested in this study free of charge.

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Comparison of methods in the recovery of nucleic acids from archival formalin-fixed paraffin-embedded autopsy tissues

Abstract

Section snippets

Sampling and laboratory work authentication

Total DNA recovery and c-Myc DNA quantitation

Total DNA, amplifiable nuclear DNA copies, and inhibition

Conclusion

Acknowledgments

Methods

Am. J. Pathol.

J. Mol. Diagn.

Biochem. Biophys. Res. Commun.

Prog. Nucleic Acid Res. Mol. Biol.

Am. J. Pathol.

Am. J. Pathol.

J. Virol. Methods

J. Mol. Diagn.

Am. J. Pathol.

J. Virol. Methods

J. Mol. Diagn.

Direct evidence of extensive diversity of HIV-1 in Kinshasa by 1960

Nature

Initial genetic characterization of the 1918 “Spanish” influenza virus

Science

Optimization of RNA extraction from FFPE tissues for expression profiling in the DASL assay

BioTechniques

On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions

Histochem. Cell Biol.

The applicability of formalin-fixed and formalin-fixed paraffin-embedded tissues in forensic DNA analysis

J. Forensic Sci.

The prevalence of Karposi’s sarcoma (KS) in the Uganda Biopsy Service before and during the AIDS era, 1965–1995

Uganda Med. J.

Evaluation of DNA extraction methods and real time PCR optimization on formalin-fixed paraffin-embedded tissues

Asian Pac. J. Cancer Prev.

The isolation of nucleic acids from fixed, paraffin-embedded tissues: which methods are useful when?

PLoS ONE

Purification and analysis of RNA from paraffin-embedded tissues

BioTechniques

Comparison of the DNA extraction methods for polymerase chain reaction amplification from formalin-fixed and paraffin-embedded tissues

Diagn. Mol. Pathol.

DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle: Heating under the influence of pH

J. Histochem. Cytochem.

DNA extraction from archival formalin-fixed, paraffin-embedded tissues: heat-induced retrieval in alkaline solution

Histochem. Cell Biol.

Extraction and amplification of DNA from formalin-fixed, paraffin-embedded tissues

Appl. Immunohistochem. Mol. Morph.

Conversions of formaldehyde-modified 2’-deoxyadenosine 5’-monophosphate in conditions modeling formalin-fixed tissue dehydration

J. Histochem. Cytochem.