Dec 13, 2024
NCI Biospecimen Evidence-Based Practices (BEBP) - Nucleic Acid Extraction from Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue
- 1National Cancer Institute
External link: https://biospecimens.cancer.gov/resources/bebp.asp
Protocol Citation: NCI Biorepositories and Biospecimen Research Branch 2024. NCI Biospecimen Evidence-Based Practices (BEBP) - Nucleic Acid Extraction from Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5r97jg1b/v1
Manuscript citation:
Greytak SR, Engel KB, Zmuda E, Casas-Silva E, Guan P, Hoadley KA, Mungall AJ, Wheeler DA, Doddapaeneni HV, Moore HM. (2018). National Cancer Institute Biospecimen Evidence-Based Practices: Harmonizing Procedures for Nucleic Acid Extraction from Formalin-Fixed, Paraffin-Embedded Tissue. Biopreservation and Biobanking. 16(4):247-250. PMID 29920119.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
This document contains guidance that is intended to facilitate the development of evidence-based standard operating procedures.
Created: May 01, 2024
Last Modified: December 13, 2024
Protocol Integer ID: 99077
Keywords: FFPE, DNA, RNA, nucleic acid, extraction, NCI, BEBP, evidence-based, tissue
Disclaimer
This document contains guidance that is intended to facilitate the development of evidence-based standard operating procedures.
Abstract
This evidence-based, expert-vetted best practice document is applicable for nucleic acid extraction from standard-size FFPE blocks containing human tissue, although such guidance may also be applicable to tissue from mammalian animal models. Specifically, recommended procedures are suitable for standard cassette-sized, non-decalcified tissue biospecimens that were fixed in 10% neutral buffered formalin (NBF) for less than 48 h before paraffin embedding. Biospecimens processed under these procedural guidelines are suitable for analysis by polymerase chain reaction (PCR), methylation analysis, Sanger sequencing and next-generation sequencing, and microarray hybridization. Much of the evidence presented in this document pertains to mRNA molecules and DNA, thus shorter RNA molecules such as microRNAs (miRNAs) may require additional consideration and optimization. This document is not applicable for analysis of biospecimens by in situ hybridization.
Attachments
NCI BEBP_Expert-vett...
1,000KB
Guidelines
9.1.1 2007 Guideline for Isolation Precautions: Preventing Transmission of Infectious Agents in Healthcare Settings (CDC, 2007): http://www.cdc.gov/hicpac/2007IP/2007isolationPrecautions.html
9.1.3 CLSI IL-28A: Quality Assurance for Design Control and Implementation of Immunohistochemistry Assays; Approved Guideline—Second Edition S Hewitt, personal communications, draft CLSI IL-28a
9.1.4 QIAGEN (June, 2012) QIAamp‱ DNA FFPE Tissue Handbook, Valencia CA
9.1.5 QIAGEN (February, 2013) RNeasy‱ FFPE Handbook, Valencia CA
9.1.6 Life Technologies (2011) Ambion RecoverAll‱ Total Nucleic Acid Isolation Kit, Carlsbad CA
9.1.7 QIAGEN (2012) AllPrep‱ DNA/RNA FFPE Handbook, Valencia CA
9.1.8 TrimGen, WaxFreeTM RNA User Manual, Sparks MD
9.1.9 Biorepositories and Biospecimen Research Branch, National Cancer Institute, National Institutes of Health. NCI Biospecimen Evidence-Based Practices, Formalin-Fixation and Paraffin Processing of Tissue Biospecimens. In Preparation.
9.1.10 Broad Institute Genomic Services (2016) Nucleic Acid Extractions: Product Data Sheet, http://genomics.broadinstitute.org/data-sheets/DTS_Extractions_1Page_5-2016.pdf
Materials
5.1 RNase removal spray or wipes
5.2 Microtome
5.3 Ice block
5.4 Pre-labeled RNase-free microcentrifuge tubes
5.5 Plastic-backed absorbent bench paper
5.6 Xylene
5.7 Ethanol
5.8 Microcentrifuge
5.9 An experimentally-validated extraction kit of the user's choice. Acceptable methods for DNA extraction include silica, magnetic bead, heating, focused-ultrasonication, resin-filter, or salting-out-based methods, but organic extraction should be avoided (See Section 7.8 in the Attached PDF for Literature Evidence). Acceptable RNA extraction methods include silica, magnetic bead, organic focused-ultrasonication or salting-out-based methods, but generally, resin-filter and heat-based methods should be avoided (See Section 7.9 in the Attached PDF for Literature Evidence).
5.10 Real-time PCR machine
5.11 Spectrophotometer
5.12 Fluorometric DNA quantification method of choice
Safety warnings
Guidelines on precautions to prevent the transmission of infectious agents should be consulted and can be used for all phases of organ/tissue dissection and handling (Reference 9.1.1).
The National Institute for Occupational Safety and Health (NIOSH) Pocket Guide to Chemical Hazards should be consulted for all steps including formalin, xylene, and other chemicals (Reference 9.1.2).
Ethics statement
Protocols developed using this Biospecimen Evidence-Based Practice may require approval by the user’s institutional review board (IRB) or an equivalent ethics committee prior to implementation.
Before start
The purpose of this document is to provide evidence-based and expert-vetted guidance on nucleic acid extraction from formalin-fixed and paraffin-embedded (FFPE) human tissues. This guidance is intended to support the development and execution of evidence-based Standard Operating Procedures (SOPs) for DNA and RNA extraction from FFPE biospecimens.
Recording of biospecimen pre-acquisition, acquisition and processing data
Recording of biospecimen pre-acquisition, acquisition and processing data
Whenever possible, extensive data should be recorded relating to preacquisition,
acquisition and processing conditions that may affect the integrity
of the biospecimen. Such data may consist of patient information (including
age, gender, diagnosis, and treatment), details relating to surgery and
biospecimen acquisition (including the use of anesthesia, warm ischemia
time, and surgical procedure and duration), and FFPE processing (including
fixation duration, temperature, embedding medium) and the duration of
block storage (Guideline 9.1.9).
Preparation of bench space
Preparation of bench space
Pre-label microcentrifuge tubes.
Clean work area and metal equipment including microtome with a xylene-free
deparaffinization reagent (See Section 8.3.2 in the Attached PDF for Expert Recommendations).
Wipe down all equipment, work area and all tools with RNase removal wipes
or spray.
Sectioning paraffin blocks
Sectioning paraffin blocks
Place paraffin blocks face-down on a cold plate for a minimum of 00:05:00 or at room temperature if blocks were stored at -20°C. The 5 min incubation may be reduced if the block has been stored under recommended conditions (See Section 7.1 in the Attached PDF for Literature Evidence). Placement of paraffin blocks on wet ice is not recommended (See Section 8.3.2 in the Attached PDF for Expert
Recommendations).
A new microtome blade should be used for each biospecimen.
Position the FFPE tissue block in the specimen clamp of the microtome.
Cut sections that are 5-10 μm thick (Guideline 9.1.4) (See Section 7.2 in the Attached PDF for Literature Evidence). Allow individual sections to roll up naturally. After discarding the first 3-4 sections, place the number of sections required to obtain the desired volume of tissue needed for extraction directly into a sterile RNase-free microfuge tube using RNase-free forceps. For reference, 1-8 sections will be required for a block with a tissue surface area of 250 mm2 (Guideline 9.1.10). However, the optimal number of sections for extraction is based on the tissue surface area and cellularity (determined by H&E staining) of a given block and the extraction kit used; thus, the number of sections or tissue area needed for extraction should be experimentally validated for each extraction kit and standardized within a specimen cohort (See Section 8.3.3 in the Attached PDF for Expert Recommendations).
Paraffin sections should be processed for extraction immediately, optimally within 24 h. While available literature is limited, evidence suggests that when necessary, sections may be stored for up to 90 days at room temperature (See Section 7.3 in the Attached PDF for Literature Evidence) or 4, -20 or -80˚C in a sealed microcentrifuge tube (See Section 8.3.4 in the Attached PDF for Expert Recommendations) for some analyses.
Deparaffinization of sections
Deparaffinization of sections
Add 1 ml of xylene to the microcentrifuge tube or slide and incubate at Room temperature for 00:10:00 to deparaffinize sections. Acceptable alternative deparaffinization methods are discussed in Sections 7.4 (Literature Evidence) and 8.3.5 (Expert Recommendations) in the Attached PDF. For DNA extraction, acceptable alternatives include use of proprietary buffers, mineral oil, lysis buffer heated to 98-120°C, focused-ultrasonication, heptane or the xylene substitute HemoClear. For RNA extraction, acceptable alternatives include use of proprietary buffers, heptane, lysis buffer heated to 95-98°C, focused-ultrasonication or the xylene substitute HemoD. Microwave heating should be avoided (See Section 7.4 in
the Attached PDF for Literature Evidence). The acceptable duration of deparaffinization will depend upon the method employed (See Section 7.5 in the Attached PDF for Literature Evidence).
Centrifuge tubes for 00:02:00 at maximum speed (approximately 16,000-20,000 x g) (Guidelines 9.1.4, 9.1.5, 9.1.6, and 9.1.7) and discard the supernatant.
Wash the pellet/slide in 100% ethanol, vortex, and centrifuge at maximum speed at room temperature. Discard the supernatant. A second ethanol wash may be necessary for some deparaffinization techniques and specimens of low quality (See Section 8.3.5 in the Attached PDF for Expert Recommendations).
Ensure the pellet is completely dry. Ethanol may be removed from the pellet by air-drying at Room temperature or with the use of a speed vacuum.
Proteinase K digestion
Proteinase K digestion
Add lysis buffer containing proteinase K at the volume and concentration specified by the extraction kit and incubate at 55-56°C for 2-48 h for DNA or for 15 min - overnight for RNA. The optimal duration of digestion is dependent upon multiple factors, including buffer composition and enzyme concentration, and must be determined experimentally. Other digestion durations and lower temperatures may also be acceptable (In the Attached PDF, see Sections 7.6 (Literature Evidence) and 8.3.6 (Expert Recommendations) for alternatives).
Heat tubes to 90°C for 10 min-2 h for DNA extraction or to 80°C for 0-15 min for RNA extraction. Optimal durations of demodification will be dependent upon the extraction kit used (See Section 7.7 in the Attached PDF for Literature Evidence). This step may be unnecessary for some analyses, in which case it can be omitted. For alternate temperatures and durations see Sections 7.7 (Literature Evidence) and 8.3.7 (Expert Recommendations) in the attached PDF.
DNA and RNA extraction
DNA and RNA extraction
Continue to DNA or RNA extraction using the method specified by the experimentally validated nucleic acid extraction kit (See Sections 7.8 and 7.9 in the Attached PDF for Literature Evidence). Acceptable methods for DNA extraction include silica, magnetic-bead, heating, resin-filter, ultrasonication or salting-out based methods, but organic extraction should be avoided (See Sections 7.8 and 8.3.8 in the Attached PDF for Literature Evidence and Expert Recommendations, respectively). Acceptable RNA extraction methods include silica, magnetic bead, organic, ultrasonication, or salting-out based methods, but generally resin filter and heat-based methods should be avoided (See Sections 7.9 and 8.3.8 in the Attached PDF for Literature Evidence and Expert Recommendations, respectively).
Post-extraction treatment
Post-extraction treatment
Remove contaminating nucleic acids by RNase treatment of DNA or DNase treatment of RNA is advised but not required (See Sections 7.10 and 8.3.9 in the Attached PDF for Literature Evidence and Expert Recommendations, respectively).
Nucleic acid quantification and quality assessment
Nucleic acid quantification and quality assessment
Quantify DNA and RNA using a fluorometric or qPCR-based method. While spectrophotometry should be avoided for DNA quantification as it overestimates yield, it can provide valuable information on DNA purity and quality. Spectrophotometry may be acceptable for RNA quantification, although inconsistent and overestimated yields have been observed with incomplete DNAse treatment (See Sections 7.11 and 8.3.10 in the Attached PDF for Literature Evidence and Expert Recommendations, respectively).
Verify DNA integrity using fit-for-purpose quality metrics. Suggested DNA quality guidelines are organized by analytical platform in Table 1 in the attached PDF (See Sections 7.12 and 8.3.11 in the Attached PDF for Literature Evidence and Expert Recommendations, respectively).
Verify RNA quality using the percentage of RNA fragments >200 nt (DV200) and/or a combination of DV200 and real-time qRT-PCR amplification of products from the 5’ and 3’ end of the transcript. RIN is not an informative method of RNA quality assessment for FFPE specimens and should be avoided. Suggested RNA quality guidelines are listed by analytical platform in Table 2 in the attached PDF (See Sections 7.13 and 8.3.12 in the Attached PDF for Literature Evidence and Expert Recommendations, respectively).
Protocol references
References considered during the development of this NCI BEBP document are listed below (also See Section 9.2 in the Attached PDF) and include hyperlinks to the PubMed abstract and NCI Biospecimen Research Database curation where applicable. References are cited within the Summaries of Literature Evidence (See Section 7.0) in the Attached PDF.
1. Bass, B.P., et al., A review of pre-analytical factors affecting molecular, protein, and morphological analysis of formalin-fixed, paraffin-embedded tissue. Arch Pathol Lab Med., 2014. 138(11): p. 1520-1530.
2. Coombs, N.J., A.C. Gough, and J.N. Primrose, Optimisation of DNA and RNA extraction from archival formalin-fixed tissue. Nucleic Acids Res, 1999. 27(16): p. e12.
3. Gall, K., et al., DNA amplification by polymerase chain reaction from brain tissues embedded in paraffin. Int J Exp Pathol, 1993. 74(4): p. 333-7.
4. Goelz, S.E., S.R. Hamilton, and B. Vogelstein, Purification of DNA from formaldehyde fixed and paraffin embedded human tissue. Biochem Biophys Res Commun, 1985. 130(1): p. 118-26.
5. Greer, C.E., C.M. Wheeler, and M.M. Manos, Sample preparation and PCR amplification from paraffin-embedded tissues. PCR Methods Appl, 1994. 3(6): p. S113-22.
6. Hewett, P.J., F. Firgaira, and A. Morley, The influence of age of template DNA derived from archival tissue on the outcome of the polymerase chain reaction. Aust N Z J Surg, 1994. 64(8): p. 558-9.
7. Jaremko, M., et al., MALDI-TOF MS and TaqMan assisted SNP genotyping of DNA isolated from formalin-fixed and paraffin-embedded tissues (FFPET). Hum Mutat, 2005. 25(3): p. 232-8.
8. Merkelbach, S., et al., Novel enzyme immunoassay and optimized DNA extraction for the detection of polymerase-chain-reaction-amplified viral DNA from paraffin-embedded tissue. Am J Pathol, 1997. 150(5): p. 1537-46.
9. Pavelic, J., et al., PCR amplification of DNA from archival specimens. A methodological approach. Neoplasma, 1996. 43(2): p. 75-81.
10. Talaulikar, D., et al., DNA amplification from formalin-fixed decalcified paraffin-embedded bone marrow trephine specimens: does the duration of storage matter? Pathology, 2008. 40(7): p. 702-6.
11. Turashvili, G., et al., Nucleic acid quantity and quality from paraffin blocks: defining optimal fixation, processing and DNA/RNA extraction techniques. Exp Mol Pathol, 2012. 92(1): p. 33-43.
12. Guyard, A., et al., DNA degrades during storage in formalin-fixed and paraffin-embedded tissue blocks. Virchows Arch, 2017.
13. Wang, G., et al., DNA amplification method tolerant to sample degradation. Genome Res, 2004. 14(11): p. 2357-66.
14. Abdueva, D., et al., Quantitative expression profiling in formalin-fixed paraffin-embedded samples by affymetrix microarrays. J Mol Diagn, 2010. 12(4): p. 409-17.
15. Beaulieu, M., et al., Analytical Performance of a qRT-PCR Assay to Detect Guanylyl Cyclase C in FFPE Lymph Nodes of Patients With Colon Cancer. Diagn Mol Pathol, 2010. 19(1): p. 20-7.
16. Ribeiro-Silva, A., H. Zhang, and S.S. Jeffrey, RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies. BMC Mol Biol, 2007. 8: p. 118.
17. Liu, H., et al., Archival fixed histologic and cytologic specimens including stained and unstained materials are amenable to RT-PCR. Diagn Mol Pathol, 2002. 11(4): p. 222-7.
18. Frank, M., et al., Global gene expression profiling of formalin-fixed paraffin-embedded tumor samples: a comparison to snap-frozen material using oligonucleotide microarrays. Virchows Arch, 2007. 450(6): p. 699-711.
19. Cronin, M., et al., Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay. Am J Pathol, 2004. 164(1): p. 35-42.
20. Bohmann, K., et al., RNA extraction from archival formalin-fixed paraffin-embedded tissue: a comparison of manual, semiautomated, and fully automated purification methods. Clin Chem, 2009. 55(9): p. 1719-27.
21. Müller, B.M., et al., Quantitative determination of estrogen receptor, progesterone receptor, and HER2 mRNA in formalin-fixed paraffin-embedded tissue--a new option for predictive biomarker assessment in breast cancer. Diagn Mol Pathol, 2011. 20(1): p. 1-10.
22. Stewart, G.D., et al., Utilizing mRNA extracted from small, archival formalin-fixed paraffin-embedded prostate samples for translational research: assessment of the effect of increasing sample age and storage temperature. Int Urol Nephrol, 2011. 43(4): p. 961-7.
23. Godfrey, T.E., et al., Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5' nuclease quantitative reverse transcription-polymerase chain reaction. J Mol Diagn, 2000. 2(2): p. 84-91.
24. Chu, W.S., et al., Ultrasound-accelerated tissue fixation/processing achieves superior morphology and macromolecule integrity with storage stability. J Histochem Cytochem, 2006. 54(5): p. 503-13.
25. Guerrero, R.B., et al., Effects of formalin fixation and prolonged block storage on detection of hepatitis C virus RNA in liver tissue. Diagn Mol Pathol, 1997. 6(5): p. 277-81.
26. Xi, Y., et al., Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples. RNA, 2007. 13(10): p. 1668-74.
27. Isola, J., et al., Analysis of changes in DNA sequence copy number by comparative genomic hybridization in archival paraffin-embedded tumor samples. Am J Pathol, 1994. 145(6): p. 1301-8.
28. Neubauer, A., et al., Analysis of gene amplification in archival tissue by differential polymerase chain reaction. Oncogene, 1992. 7(5): p. 1019-25.
29. Forsthoefel, K.F., et al., Optimization of DNA extraction from formalin-fixed tissue and its clinical application in Duchenne muscular dystrophy. Am J Clin Pathol, 1992. 98(1): p. 98-104.
30. Rogers, B.B., et al., Analysis of DNA in fresh and fixed tissue by the polymerase chain reaction. Am J Pathol, 1990. 136(3): p. 541-8.
31. Doleshal, M., et al., Evaluation and validation of total RNA extraction methods for microRNA expression analyses in formalin-fixed, paraffin-embedded tissues. J Mol Diagn, 2008. 10(3): p. 203-11.
32. Roberts, L., et al., Identification of methods for use of formalin-fixed, paraffin-embedded tissue samples in RNA expression profiling. Genomics, 2009. 94(5): p. 341-8.
33. Whetsell, L., et al., Polymerase chain reaction microanalysis of tumors from stained histological slides. Oncogene, 1992. 7(11): p. 2355-61.
34. Gjerdrum, L.M., et al., The influence of immunohistochemistry on mRNA recovery from microdissected frozen and formalin-fixed, paraffin-embedded sections. Diagn Mol Pathol, 2004. 13(4): p. 224-33.
35. Wu, L., et al., Extraction and amplification of DNA from formalin-fixed, paraffin-embedded tissues. Appl Immunohistochem Mol Morphol, 2002. 10(3): p. 269-74.
36. Steinau, M., S.S. Patel, and E.R. Unger, Efficient DNA extraction for HPV genotyping in formalin-fixed, paraffin-embedded tissues. J Mol Diagn, 2011. 13(4): p. 377-81.
37. Atanesyan, L., et al., Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA. Am J Clin Pathol, 2017. 147(1): p. 60-68.
38. Gilbert, M.T., et al., The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when? PLoS One, 2007. 2(6): p. e537.
39. Potluri, K., et al., Genomic DNA extraction methods using formalin-fixed paraffin-embedded tissue. Anal Biochem, 2015. 486: p. 17-23.
40. Kikuchi, A., et al., Improved protocol for extraction of genomic DNA from formalin-fixed paraffin-embedded tissue samples without the use of xylene. Clin Chem Lab Med, 2016. 54(12): p. e375-e377.
41. Heikal, N., R.H. Nussenzveig, and A.M. Agarwal, Deparaffinization with mineral oil: a simple procedure for extraction of high-quality DNA from archival formalin-fixed paraffin-embedded samples. Appl Immunohistochem Mol Morphol, 2014. 22(8): p. 623-6.
42. Banerjee, S.K., et al., Microwave-based DNA extraction from paraffin-embedded tissue for PCR amplification. Biotechniques, 1995. 18(5): p. 768-70, 772-3.
43. Sato, Y., et al., Comparison of the DNA extraction methods for polymerase chain reaction amplification from formalin-fixed and paraffin-embedded tissues. Diagn Mol Pathol, 2001. 10(4): p. 265-71.
44. Krafft, A.E., et al., Optimization of the Isolation and Amplification of RNA From Formalin-fixed, Paraffin-embedded Tissue: The Armed Forces Institute of Pathology Experience and Literature Review. Mol Diagn, 1997. 2(3): p. 217-230.
45. Okayama, N., et al., The importance of evaluation of DNA amplificability in KRAS mutation testing with dideoxy sequencing using formalin-fixed and paraffin-embedded colorectal cancer tissues. Jpn J Clin Oncol, 2011. 41(2): p. 165-71.
46. Chung, J.Y., T. Braunschweig, and S.M. Hewitt, Optimization of recovery of RNA from formalin-fixed, paraffin-embedded tissue. Diagn Mol Pathol, 2006. 15(4): p. 229-36.
47. Kling, T., et al., Validation of the MethylationEPIC BeadChip for fresh-frozen and formalin-fixed paraffin-embedded tumours. Clin Epigenetics, 2017. 9: p. 33.
48. Espinal, A.C., et al., A methodological study of genome-wide DNA methylation analyses using matched archival formalin-fixed paraffin embedded and fresh frozen breast tumors. Oncotarget, 2017. 8(9): p. 14821-14829.
49. Bak, S.T., et al., Evaluating the Feasibility of DNA Methylation Analyses Using Long-Term Archived Brain Formalin-Fixed Paraffin-Embedded Samples. Mol Neurobiol, 2016.
50. Min, J., et al., Methylation Levels of LINE-1 As a Useful Marker for Venous Invasion in Both FFPE and Frozen Tumor Tissues of Gastric Cancer. Mol Cells, 2017. 40(5): p. 346-354.
51. Wen, X., et al., Improved results of LINE-1 methylation analysis in formalin-fixed, paraffin-embedded tissues with the application of a heating step during the DNA extraction process. Clin Epigenetics, 2017. 9: p. 1.
52. Shao, D., et al., A targeted next-generation sequencing method for identifying clinically relevant mutation profiles in lung adenocarcinoma. Sci Rep, 2016. 6: p. 22338.
53. Einaga, N., et al., Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation. PLoS One, 2017. 12(5): p. e0176280.
54. Kim, S., et al., Deamination Effects in Formalin-Fixed, Paraffin-Embedded Tissue Samples in the Era of Precision Medicine. J Mol Diagn, 2017. 19(1): p. 137-146.
55. Hedegaard, J., et al., Next-generation sequencing of RNA and DNA isolated from paired fresh-frozen and formalin-fixed paraffin-embedded samples of human cancer and normal tissue. PLoS One, 2014. 9(5): p. e98187.
56. Roy-Chowdhuri, S., et al., Concurrent fine needle aspirations and core needle biopsies: a comparative study of substrates for next-generation sequencing in solid organ malignancies. Mod Pathol, 2017. 30(4): p. 499-508.
57. Biesaga, B., et al., HPV16 detection by qPCR method in relation to quantity and quality of DNA extracted from archival formalin fixed and paraffin embedded head and neck cancer tissues by three commercially available kits. J Virol Methods, 2016. 236: p. 157-63.
58. Ayatollahi, H., et al., The effect of deoxyribonucleic acid extraction methods from lymphoid tissue on the purity, content, and amplifying ability. Niger Med J, 2016. 57(4): p. 199-203.
59. Vojtechova, Z., et al., Comparison of the miRNA expression profiles in fresh frozen and formalin-fixed paraffin-embedded tonsillar tumors. PLoS One, 2017. 12(6): p. e0179645.
60. Kolbert, C.P., et al., Multi-platform analysis of microRNA expression measurements in RNA from fresh frozen and FFPE tissues. PLoS One, 2013. 8(1): p. e52517.
61. Lu, Y., et al., RNA Extracted from Formalin-Fixed, Paraffin-Embedded Renal Biopsy Biospecimens: An Evaluation of Alternative Extraction Kits and the Effects of Storage Time. Biopreserv Biobank, 2017. 15(4): p. 396-398.
62. Guo, Y., et al., RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Specimens for Gene Expression Quantification and Data Mining. Int J Genomics, 2016. 2016: p. 9837310.
63. Tyekucheva, S., et al., Comparing Platforms for Messenger RNA Expression Profiling of Archival Formalin-Fixed, Paraffin-Embedded Tissues. J Mol Diagn, 2015. 17(4): p. 374-81.
64. Belder, N., et al., From RNA isolation to microarray analysis: Comparison of methods in FFPE tissues. Pathol Res Pract, 2016. 212(8): p. 678-85.
65. Eikrem, O., et al., Transcriptome Sequencing (RNAseq) Enables Utilization of Formalin-Fixed, Paraffin-Embedded Biopsies with Clear Cell Renal Cell Carcinoma for Exploration of Disease Biology and Biomarker Development. PLoS One, 2016. 11(2): p. e0149743.
66. Arzt, L., et al., Evaluation of formalin-free tissue fixation for RNA and microRNA studies. Exp Mol Pathol, 2011. 91(2): p. 490-495.
67. Bonin, S., et al., Multicentre validation study of nucleic acids extraction from FFPE tissues. Virchows Arch, 2010. 457(3): p. 309-17.
68. Graw, S., et al., Robust gene expression and mutation analyses of RNA-sequencing of formalin-fixed diagnostic tumor samples. Sci Rep, 2015. 5: p. 12335.
69. Loudig, O., et al., Evaluation and Adaptation of a Laboratory-Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens. Int J Mol Sci, 2017. 18(3): p. 627.
70. Frank, T.S., S.M. Svoboda-Newman, and E.D. Hsi, Comparison of methods for extracting DNA from formalin-fixed paraffin sections for nonisotopic PCR. Diagn Mol Pathol, 1996. 5(3): p. 220-4.
71. Masuda, N., et al., Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples. Nucleic Acids Res, 1999. 27(22): p. 4436-43.
72. Shi, S.R., et al., 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, 2002. 50(8): p. 1005-11.
73. Shi, S.R., et al., DNA extraction from archival formalin-fixed, paraffin-embedded tissues: heat-induced retrieval in alkaline solution. Histochem Cell Biol, 2004. 122(3): p. 211-8.
74. Torrente, M.C., et al., DNA extraction from formalin-fixed laryngeal biopsies: Comparison of techniques. Acta Otolaryngol, 2011. 131(3): p. 330-3.
75. Hamatani, K., et al., Improved RT-PCR amplification for molecular analyses with long-term preserved formalin-fixed, paraffin-embedded tissue specimens. J Histochem Cytochem, 2006. 54(7): p. 773-80.
76. Oberli, A., et al., Expression profiling with RNA from formalin-fixed, paraffin-embedded material. BMC Med Genomics, 2008. 1: p. 9.
77. Wehmas, L.C., et al., Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples. Toxicol Sci, 2017.
78. Huijsmans, C.J., et al., Comparative analysis of four methods to extract DNA from paraffin-embedded tissues: effect on downstream molecular applications. BMC Res Notes, 2010. 3: p. 239.
79. Jacobs, S., et al., Genome-wide, high-resolution detection of copy number, loss of heterozygosity, and genotypes from formalin-fixed, paraffin-embedded tumor tissue using microarrays. Cancer Res, 2007. 67(6): p. 2544-51.
80. Schweiger, M.R., et al., Genome-wide massively parallel sequencing of formaldehyde fixed-paraffin embedded (FFPE) tumor tissues for copy-number- and mutation-analysis. PLoS One, 2009. 4(5): p. e5548.
81. Sam, S.S., et al., Automation of genomic DNA isolation from formalin-fixed, paraffin-embedded tissues. Pathol Res Pract, 2012. 208(12): p. 705-7.
82. Khokhar, S.K., et al., Evaluation of Maxwell(‱) 16 for automated DNA extraction from whole blood and formalin-fixed paraffin embedded (FFPE) tissue. Clin Chem Lab Med, 2012. 50(2): p. 267-272.
83. Funabashi, K.S., et al., DNA extraction and molecular analysis of non-tumoral liver, spleen, and brain from autopsy samples: The effect of formalin fixation and paraffin embedding. Pathol Res Pract, 2012. 208(10): p. 584-91.
84. Dedhia, P., et al., Evaluation of DNA extraction methods and real time PCR optimization on formalin-fixed paraffin-embedded tissues. Asian Pac J Cancer Prev, 2007. 8(1): p. 55-9.
85. Little, S.E., et al., Array CGH using whole genome amplification of fresh-frozen and formalin-fixed, paraffin-embedded tumor DNA. Genomics, 2006. 87(2): p. 298-306.
86. Patel, P.G., et al., Reliability and performance of commercial RNA and DNA extraction kits for FFPE tissue cores. PLoS One, 2017. 12(6): p. e0179732.
87. Ludyga, N., et al., Nucleic acids from long-term preserved FFPE tissues are suitable for downstream analyses. Virchows Arch, 2012. 460(2): p. 131-40.
88. Kalmár, A., et al., Comparison of Automated and Manual DNA Isolation Methods for DNA Methylation Analysis of Biopsy, Fresh Frozen, and Formalin-Fixed, Paraffin-Embedded Colorectal Cancer Samples. J Lab Autom, 2015. 20(6): p. 642-651.
89. Heydt, C., et al., Comparison of pre-analytical FFPE sample preparation methods and their impact on massively parallel sequencing in routine diagnostics. PLoS One, 2014. 9(8): p. e104566.
90. Cao, W., et al., Comparison of methods for DNA extraction from paraffin-embedded tissues and buccal cells. Cancer Detect Prev, 2003. 27(5): p. 397-404.
91. Boeckx, C., et al., Expression analysis on archival material: comparison of 5 commercially available RNA isolation kits for FFPE material. Diagn Mol Pathol, 2011. 20(4): p. 203-11.
92. Sharma, M., et al., Ribonucleic acid extraction from archival formalin fixed paraffin embedded myocardial tissues for gene expression and pathogen detection. J Clin Lab Anal, 2012. 26(4): p. 279-85.
93. Kashofer, K., et al., Quality control of RNA preservation and extraction from paraffin-embedded tissue: implications for rt-PCR and microarray analysis. PLoS One, 2013. 8(7): p. e70714.
94. Fedorowicz, G., et al., Microarray analysis of RNA extracted from formalin-fixed, paraffin-embedded and matched fresh-frozen ovarian adenocarcinomas. BMC Med Genomics, 2009. 2: p. 23.
95. Okello, J.B., et al., Comparison of methods in the recovery of nucleic acids from archival formalin-fixed paraffin-embedded autopsy tissues. Anal Biochem, 2010. 400(1): p. 110-7.
96. Sanchez, I., et al., How Severely Is DNA Quantification Hampered by RNA Co-extraction? Biopreserv Biobank, 2015. 13(5): p. 320-4.
97. Kapp, J.R., et al., Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial. J Clin Pathol, 2015. 68(2): p. 111-8.
98. Simbolo, M., et al., DNA qualification workflow for next generation sequencing of histopathological samples. PLoS One, 2013. 8(6): p. e62692.
99. Sah, S., et al., Functional DNA quantification guides accurate next-generation sequencing mutation detection in formalin-fixed, paraffin-embedded tumor biopsies. Genome Med, 2013. 5(8): p. 77.
100. Kumar, D., et al., Quantification of DNA Extracted from Formalin Fixed Paraffin-Embeded Tissue Comparison of Three Techniques: Effect on PCR Efficiency. J Clin Diagn Res, 2016. 10(9): p. BC01-BC03.
101. Serizawa, M., et al., The efficacy of uracil DNA glycosylase pretreatment in amplicon-based massively parallel sequencing with DNA extracted from archived formalin-fixed paraffin-embedded esophageal cancer tissues. Cancer Genet, 2015. 208(9): p. 415-27.
102. Abramovitz, M., et al., Optimization of RNA extraction from FFPE tissues for expression profiling in the DASL assay. BioTechniques, 2008. 44(3): p. 417-423.
103. Wang, F., et al., DNA degradation test predicts success in whole-genome amplification from diverse clinical samples. J Mol Diagn, 2007. 9(4): p. 441-51.
104. van Beers, E.H., et al., A multiplex PCR predictor for aCGH success of FFPE samples. Br J Cancer, 2006. 94(2): p. 333-7.
105. Bettoni, F., et al., A straightforward assay to evaluate DNA integrity and optimize next-generation sequencing for clinical diagnosis in oncology. Exp Mol Pathol, 2017. 103(3): p. 294-299.
106. Betsou, F., et al., Assays for Qualification and Quality Stratification of Clinical Biospecimens Used in Research: A Technical Report from the ISBER Biospecimen Science Working Group. Biopreserv Biobank, 2016. 14(5): p. 398-409.
107. Penland, S.K., et al., RNA expression analysis of formalin-fixed paraffin-embedded tumors. Lab Invest, 2007. 87(4): p. 383-91.
108. Waddell, N., et al., Gene expression profiling of formalin-fixed, paraffin-embedded familial breast tumours using the whole genome-DASL assay. J Pathol, 2010. 221(4): p. 452-61.
109. Hall, J.S., et al., Exon-array profiling unlocks clinically and biologically relevant gene signatures from formalin-fixed paraffin-embedded tumour samples. Br J Cancer, 2011. 104(6): p. 971-81.
110. Duenwald, S., et al., Development of a microarray platform for FFPET profiling: application to the classification of human tumors. J Transl Med, 2009. 7: p. 65.
111. Dang, J., et al., Development of a robust DNA quality and quantity assessment qPCR assay for targeted next-generation sequencing library preparation. Int J Oncol, 2016. 49(4): p. 1755-65.
Acknowledgements
We thank Dr, Erik Zmuda (Nationwide Children's Hospital, US), Dr. Fay Betsou (IBBL, Luxembourg), Dr. Jakob Hedegaard (MOMA, Denmark), Dr. William Mathieson (IBBL, Luxembourg), and Dr. Geraldine Thomas (Imperial College, England) for their participation on the expert panel and their insightful recommendations.