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