A Successful Approach to Extracted Human mtDNA and Amplify some Genes from FFPE Samples

Nisreen Abdulkadhim Hanoush; Ahmed  E.Dhamad

doi:10.31185/wjps.551

Authors

Nisreen Abdulkadhim Hanoush Department of Nurrsing Techniques, Technical Institute Kut, Middle Technical University, IRAQ
Ahmed E.Dhamad Department of Biology, College of Science, Wasit University, IRAQ

DOI:

https://doi.org/10.31185/wjps.551

Keywords:

form FFPE sample, mtDNA, Conventional PCR, Breast cancer,

Abstract

Tissues that have been paraffin-embedded and fixed in formalin (FFPE) are a significant diagnostic resource for molecular and pathophysiological methods of cancer and numerous other illnesses. The most difficult obstacle is obtaining high-quality of DNA from FFPE tissues, especially mitochondrial DNA (mtDNA), as formalin fixation significantly compromises the integrity of DNA. To extract mtDNA, the Geneaid gSYNCTM DNA kit (cat#GS100) was used with fifty breast cancer tissues (block). To check the mtDNA quality, specific primers for the following genes, ATP6 gene (8527-9024) and D-Loop region (256-16277), were used to run a PCR reaction. Our data showed that the mtDNA was successfully extracted with high quantity and quality. The two genes were amplified via PCR with very high rate (97% average). In conclusion, the method described here has many possibilities for applications in various molecular research projects involving FFPET substances. It can be used to obtain a high quantity and quality of mtDNA from FFPE samples for different molecular diagnostic and research methods such as PCR, qPCR, Sanger sequencing, and whole genome sequencing.

References

Q. Guo, E. Lakatos, I. A. Bakir, K. Curtius, T. A. Graham, and V. Mustonen, "The mutational signatures of formalin fixation on the human genome," Nature Communications, vol. 13, no. 1, p. 4487, 2022.

W. Mathieson and G. Thomas, "Using FFPE tissue in genomic analyses: advantages, disadvantages and the role of biospecimen science," Current Pathobiology Reports, vol. 7, pp. 35-40, 2019.

K. Zhou et al., "A novel next-generation sequencing–based approach for concurrent detection of mitochondrial DNA copy number and mutation," The Journal of Molecular Diagnostics, vol. 22, no. 12, pp. 1408-1418, 2020.

M. Mirmomeni, S. Sajjadi Majd, S. Sisakhtnezhad, and F. Doranegard, "Comparison of the three methods for DNA extraction from paraffin-embedded tissues," Journal of Biological Sciences, vol. 10, no. 3, pp. 261-266, 2010.

K. Vitošević, M. Todorović, Ž. Slović, T. Varljen, S. Matić, and D. Todorović, "DNA isolated from formalin-fixed paraffin-embedded healthy tissue after 30 years of storage can be used for forensic studies," Forensic Science, Medicine and Pathology, vol. 17, pp. 47-57, 2021.

P. Decruyenaere, K. Verniers, F. Poma-Soto, J. Van Dorpe, F. Offner, and J. Vandesompele, "RNA extraction method impacts quality metrics and sequencing results in formalin-fixed, paraffin-embedded tissue samples," Laboratory Investigation, vol. 103, no. 2, p. 100027, 2023.

B. P. Bass, K. B. Engel, S. R. Greytak, and H. M. Moore, "A review of preanalytical factors affecting molecular, protein, and morphological analysis of formalin-fixed, paraffin-embedded (FFPE) tissue: how well do you know your FFPE specimen?" Archives of pathology and laboratory medicine, vol. 138, no. 11, pp. 1520-1530, 2014.

L. Li, L. Chen, J. Li, W. Zhang, Y. Liao, J. Chen, and Z. Sun, "Correlational study on mitochondrial DNA mutations as potential risk factors in breast cancer," Oncotarget, vol. 7, no. 21, p. 31270, 2016.

A. Talei, M. Akrami, M. Mokhtari, and S. Tahmasebi, "Surgical and clinical pathology of breast diseases," Histopathology-Reviews and Recent Advances, 2012.

X. H. Gao et al., "Comparison of fresh frozen tissue with formalin-fixed paraffin-embedded tissue for mutation analysis using a multi-gene panel in patients with colorectal cancer," Frontiers in oncology, vol. 10, p. 310, 2020.

S. M. Hickey et al., "Fluorescence microscopy—an outline of hardware, biological handling, and fluorophore considerations," Cells, vol. 11, no. 1, p. 35, 2021.

S. Aviel-Ronen, C. Qi Zhu, B. P. Coe, N. Liu, S. K. Watson, W. L. Lam, and M. S. Tsao, "Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues," BMC genomics, vol. 7, pp. 1-10, 2006.

I. Oscorbin and M. Filipenko, "Bst polymerase—a humble relative of Taq polymerase," Computational and Structural Biotechnology Journal, 2023.

P. Dedhia, S. Tarale, G. Dhongde, R. Khadapkar, and B. Das, "Evaluation of DNA extraction methods and real time PCR optimization on formalin-fixed paraffin-embedded tissues," Asian Pacific Journal of Cancer Prevention, vol. 8, no. 1, p. 55, 2007.

S. Bonin, F. Petrera, B. Niccolini, and G. Stanta, "PCR analysis in archival postmortem tissues," Molecular Pathology, vol. 56, no. 3, p. 184, 2003.

S. S. Khan et al., "Evaluation and Comparison of Genomic DNA Extraction Methods and PCR Optimization on Archival Formalin-Fixed and Paraffin-Embedded Tissues of Oral Squamous Cell Carcinoma," Diagnostics, vol. 12, no. 5, p. 1219, 2022.

M. T. P. Gilbert et al., "The isolation of nucleic acids from fixed, paraffin-embedded tissues–which methods are useful when?" PloS one, vol. 2, no. 6, p. e537, 2007.

S. Bonin and G. Stanta, "Nucleic acid extraction methods from fixed and paraffin-embedded tissues in cancer diagnostics," Expert Review of Molecular Diagnostics, vol. 13, no. 3, pp. 271-282, 2013.

P. G. Patel et al., "Preparation of formalin-fixed paraffin-embedded tissue cores for both RNA and DNA extraction," JoVE (Journal of Visualized Experiments), no. 114, p. e54299, 2016.

L. Grzybowska-Szatkowska, B. Ślaska, J. Rzymowska, A. Brzozowska, and B. Floriańczyk, "Novel mitochondrial mutations in the ATP6 and ATP8 genes in patients with breast cancer," Molecular medicine reports, vol. 10, no. 4, pp. 1772-1778, 2014.

Y.-h. Jang and K.-i. Lim, "Recent advances in mitochondria-targeted gene delivery," Molecules, vol. 23, no. 9, p. 2316, 2018.

P. Kozakiewicz et al., "Mitochondrial DNA changes in respiratory complex I genes in brain gliomas," Biomedicines, vol. 11, no. 4, p. 1183, 2023.

M. M. Nass and S. Nass, "Intramitochondrial fibers with DNA characteristics: I. Fixation and electron staining reactions," The Journal of cell biology, vol. 19, no. 3, pp. 593-611, 1963.

N. A. Bonekamp and N.-G. Larsson, "SnapShot: mitochondrial nucleoid," Cell, vol. 172, no. 1, pp. 388-388. e1, 2018.

R. Filograna, M. Mennuni, D. Alsina, and N. G. Larsson, "Mitochondrial DNA copy number in human disease: the more the better?," FEBS letters, vol. 595, no. 8, pp. 976-1002, 2021.

C. M. Gustafsson, M. Falkenberg, and N.-G. Larsson, "Maintenance and expression of mammalian mitochondrial DNA," Annual review of biochemistry, vol. 85, no. 1, pp. 133-160, 2016.

A. L. Smith, J. C. Whitehall, and L. C. Greaves, "Mitochondrial DNA mutations in ageing and cancer," Molecular Oncology, vol. 16, no. 18, pp. 3276-3294, 2022.

P. Kowalczyk et al., "Mitochondrial oxidative stress—a causative factor and therapeutic target in many diseases," International Journal of Molecular Sciences, vol. 22, no. 24, p. 13384, 2021.

A. Amorim, T. Fernandes, and N. Taveira, "Mitochondrial DNA in human identification: a review," PeerJ, vol. 7, p. e7314, 2019.

S. Santos, D. Sá, E. Bastos, H. Guedes-Pinto, I. Gut, F. Gärtner, and R. Chaves, "An efficient protocol for genomic DNA extraction from formalin-fixed paraffin-embedded tissues," Research in veterinary science, vol. 86, no. 3, pp. 421-426, 2009.