Iranian Journal of Pathology

An Efficient and Easy- to- Use Method for Extraction of H. pylori DNA from Archival Formalin-Fixed and Paraffin-Embedded Gastric Tissues

Document Type : Original Research

Authors

Parastoo Saniee 1
Paria Ghadersoltani 1
Masoumeh Noroozpour 1
Alireza Sadjadi 2

1 Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University G.C, Tehran, Iran

2 Digestive Oncology Research Center, Digestive Diseases Research Institute, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran

https://doi.org/10.30699/ijp.2023.562113.2974
Abstract
Background & Objective: Formalin-fixed paraffin-embedded archived tissues are useful for the genetic analyses and assessment of some patients’ disease history, including infectious diseases. However, there is no established protocol for extracting bacterial DNA from the archived specimens. In this study DNA was extracted from the archived H. pylori-positive gastric biopsies by some modifications applied to the previously published protocols. The quality of the extracted DNA was assessed by amplifying H. pylori-specific 16S rRNA gene.
Methods: Fifty H. pylori-positive gastric biopsies obtained, fixed, and embedded in paraffin blocks during 2002-2008 were recruited. After paraffin removal, simultaneous proteinase K treatment and mechanical disruption using glass beads were used for the digestion of gastric tissues. DNA extraction was performed by adding one step of phenol treatment and two steps of incubation to the conventional phenol-chloroform method. The quantity and quality of the extracted DNA samples were assessed. Also, PCR was performed using primers specific for the H. pylori-specific 16S rRNA.
Results: The electrophoresis showed that intact DNAs were recovered from all biopsy samples. Amplification of the PCR products with the size of 519bp confirmed the presence of H. pylori-specific 16S rRNA gene in all the biopsies.
Conclusion: A 100% success rate for the amplification of H. pylori-specific 16S rRNA gene was achieved from all the samples. In this regard, the designed modified method resulted in the effective removal of interfering contaminations and enhanced the quality of the extracted bacterial DNA from the archived tissues. These modifications may contribute to better extraction of the intact DNA from different bacteria present in human tissues.

Keywords

DNA extraction
Paraffin-embedded tissue
H. pylori

Subjects

  1. Aviel-Ronen S, Qi Zhu C, Coe BP, Liu N, Watson SK, Lam WL, et al. Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues. BMC Genom. 2006;7(1):1-10. [DOI:10.1186/1471-2164-7-312] [PMID]
  2. Gilbert MT, Sanchez JJ, Haselkorn T, Jewell LD, Lucas SB, Van Marck E, et al. Multiplex PCR with minisequencing as an effective high‐throughput SNP typing method for formalin‐fixed tissue. Electrophoresis. 2007;28(14):2361-7. [DOI:10.1002/elps.200600589] [PMID]
  3. Li J, Smyth P, Cahill S, Denning K, Flavin R, Aherne S, et al. Improved RNA quality and TaqMan® Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials. Bmc Biotechnol. 2008;8(1):1-11. [DOI:10.1186/1472-6750-8-10] [PMID]
  4. Srinivasan M, Sedmak D, Jewell S. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol. 2002;161(6):1961-71. [DOI:10.1016/S0002-9440(10)64472-0] [PMID]
  5. Pikor LA, Enfield KS, Cameron H, Lam WL. DNA extraction from paraffin embedded material for genetic and epigenetic analyses. J Vis Exp. 2011(49):e2763. [DOI:10.3791/2763-v] [PMID]
  6. Gillio‐Tos A, De Marco L, Fiano V, Garcia‐Bragado F, Dikshit R, Boffetta P, et al. Efficient DNA extraction from 25‐year‐old paraffin‐embedded tissues: study of 365 samples. Pathology. 2007;39(3):345-8. [DOI:10.1080/00313020701329757] [PMID]
  7. Greer C, Wheeler C, Manos M. Sample preparation and PCR amplification from paraffin-embedded. Genom Res. 1994;3:S113-S22. [DOI:10.1101/gr.3.6.S113] [PMID]
  8. Quach N, Goodman MF, Shibata D. In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR. BMC Clin Pathol. 2004;4(1):1-5. [DOI:10.1186/1472-6890-4-1] [PMID]
  9. Greer CE, Lund JK, Manos M. PCR amplification from paraffin-embedded tissues: recommendations on fixatives for long-term storage and prospective studies. Genom Res. 1991;1(1):46-50. [DOI:10.1101/gr.1.1.46] [PMID]
  10. Gilbert MTP, Haselkorn T, Bunce M, Sanchez JJ, Lucas SB, Jewell LD, et al. The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when? PloS One. 2007;2(6):e537. [DOI:10.1371/journal.pone.0000537] [PMID]
  11. Tang W, David FB, Wilson MM, Barwick BG, Leyland-Jones BR, Bouzyk MM. DNA extraction from formalin-fixed, paraffin-embedded tissue. Cold Spring Harb Protoc. 2009;2009(2):pdb. prot5138. [DOI:10.1101/pdb.prot5138] [PMID]
  12. Santos MC, Saito CP, Line SR. Extraction of genomic DNA from paraffin-embedded tissue sections of human fetuses fixed and stored in formalin for long periods. Pathol Res Pract. 2008;204(9):633-6. [DOI:10.1016/j.prp.2008.04.005] [PMID]
  13. Fan H, Gulley ML. DNA extraction from paraffin-embedded tissues. Molecular pathology protocols: Springer; 2001. p. 1-4. [DOI:10.1385/1-59259-081-0:1] [PMID]
  14. Gouveia GR, Ferreira SC, Sabino EC, Siqueira SAC, Pereira J. Comparação de três protocolos distintos para extração de RNA de amostras fixadas em formalina e emblocadas em parafina. J Brasileiro Patol Medi Lab. 2011;47(6):649-54. [DOI:10.1590/S1676-24442011000600012]
  15. Coura R, Prolla J, Meurer L, Ashton-Prolla P. An alternative protocol for DNA extraction from formalin fixed and paraffin wax embedded tissue. J Clin Pathol. 2005;58(8):894-5. [DOI:10.1136/jcp.2004.021352] [PMID]
  16. Coombs N, Gough A, Primrose J. Optimisation of DNA and RNA extraction from archival formalin-fixed tissue. Nucleic Acids Res. 1999;27(16):e12-i-e-iii. [DOI:10.1093/nar/27.16.e12] [PMID]
  17. Bonin S, Petrera F, Niccolini B, Stanta G. PCR analysis in archival postmortem tissues. Mol Pathol. 2003;56(3):184. [DOI:10.1136/mp.56.3.184] [PMID]
  18. Lehmann U, Kreipe H. Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies. Methods. 2001;25(4):409-18. [DOI:10.1006/meth.2001.1263] [PMID]
  19. Cruz-Flores R, López-Carvallo JA, Cáceres-Martínez J, Dhar AK. Microbiome analysis from formalin-fixed paraffin-embedded tissues: Current challenges and future perspectives. J Microbiol Methods. 2022;196:106476. [DOI:10.1016/j.mimet.2022.106476] [PMID]
  20. Helmersen K, Aamot HV. DNA extraction of microbial DNA directly from infected tissue: an optimized protocol for use in nanopore sequencing. Sci Rep. 2020;10(1):1-7. [DOI:10.1038/s41598-020-59957-6] [PMID]
  21. Mirmomeni M, Sajjadi Majd S, Sisakhtnezhad S, Doranegard F. Comparison of the three methods for DNA extraction from paraffin-embedded tissues. J Biol Sci. 2010;10(3):261-6. [DOI:10.3923/jbs.2010.261.266]
  22. Isola J, DeVries S, Chu L, Ghazvini S, Waldman F. Analysis of changes in DNA sequence copy number by comparative genomic hybridization in archival paraffin-embedded tumor samples. Am J Pathol. 1994;145(6):1301.
  23. Sadeghipour A, Babaheidarian P. Making formalin-fixed, paraffin embedded blocks. Biobanking. 2019:253-68. [DOI:10.1007/978-1-4939-8935-5_22] [PMID]
  24. Hewitt SM, Lewis FA, Cao Y, Conrad RC, Cronin M, Danenberg KD, et al. Tissue handling and specimen preparation in surgical pathology: issues concerning the recovery of nucleic acids from formalin-fixed, paraffin-embedded tissue. Arch Pathol Lab Med. 2008;132(12):1929-35. [DOI:10.5858/132.12.1929] [PMID]
  25. Sambrook J, Russell D. Molecular cloning: A Laboratory manual. New York: Cold Spring Harbor Laboratory Press 2001.
  26. Lu Y, Redlinger TE, Avitia R, Galindo A, Goodman K. Isolation and genotyping of Helicobacter pylori from untreated municipal wastewater. Applied and Environ Microbiol. 2002;68(3):1436-9. [PMID] [DOI:10.1128/AEM.68.3.1436-1439.2002]
  27. Septimus EJ, Schweizer ML. Decolonization in prevention of health care-associated infections. Clin Microbiol Rev. 2016;29(2):201-22. [DOI:10.1128/CMR.00049-15] [PMID]
  28. Septimus E, Weinstein RA, Perl TM, Goldmann DA, Yokoe DS. Approaches for preventing healthcare-associated infections: go long or go wide? Infect Control Hospital Epidemiolo. 2014;35(7):797-801. [DOI:10.1086/676535] [PMID]
  29. Huijsmans CJ, Damen J, van der Linden JC, Savelkoul PH, Hermans MH. Comparative analysis of four methods to extract DNA from paraffin-embedded tissues: effect on downstream molecular applications. BMC Res Notes. 2010;3(1):1-9. [DOI:10.1186/1756-0500-3-239] [PMID]
  30. Banerjee S. Microwave-DNA extraction from paraffin-embedded tissue for PCR amplification. Biotechnology. 1995;18:768.
Iranian Journal of Pathology
Volume 18, Issue 4
Autumn 2023
Pages 410-414

  • Receive Date 02 November 2022
  • Revise Date 10 July 2023
  • Accept Date 09 July 2023

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