Optimization of DNA isolation method from Formalin-Fixed-Paraffin-Embedded tissues (FFPE) and comparative performance of four different Polymerase Chain Reaction (PCR) kits

Almira Zada; Puspasari S. Susanto; Raden L. Putri; Edhyana K. Sahiratmadja; Bethy S. Hernowo; Andri Rezano; Yunia Sribudiani

doi:10.14715/cmb/2018.64.13.8

Issue

Vol. 64 No. 13: Issue 13

Issue Published : November 2, 2018

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Optimization of DNA isolation method from Formalin-Fixed-Paraffin-Embedded tissues (FFPE) and comparative performance of four different Polymerase Chain Reaction (PCR) kits

https://doi.org/10.14715/cmb/2018.64.13.8

Almira Zada

Department of Biomedical Sciences, Division of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia

Puspasari S. Susanto

Research Center of Medical Genetics, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia

Raden L. Putri

Genetics and Molecular Biology Laboratory, Faculty of Medicine, Universitas Padjadjaran, Bandung 40161, Indonesia

Edhyana K. Sahiratmadja

Department of Biomedical Sciences, Division of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia

Bethy S. Hernowo

Department of Pathology Anatomy, Dr. Hasan Sadikin General Hospital, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia

Andri Rezano

Department of Biomedical Sciences, Division of Cell Biology, Faculty of Medicine, Universitas Padjadjaran, Bandung, West Java, Indonesia

http://orcid.org/0000-0003-2550-8794

Yunia Sribudiani

Department of Biomedical Sciences, Division of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia

http://orcid.org/0000-0003-1985-3292

Corresponding Author(s) : Yunia Sribudiani

y.sribudiani@unpad.ac.id

Cellular and Molecular Biology, Vol. 64 No. 13: Issue 13
Article Published : October 30, 2018

Abstract

The tissue sample may have important genetic information in diagnostic, prognostic and counselling issues. Formalin-Fixed-Paraffin-Embedded (FFPE) is a routine method for preserving tissues. However, DNA isolated from FFPE tissue is often difficult to be amplified in PCR due to fragmentation and DNA-protein crosslinks. This study aimed to optimize the DNA isolation method from FFPE tissue and compare the performance of four different PCR ready-to-use kits. Genomic DNA was isolated from FFPE tissue colon of Short-segment Hirschsprung (S-HSCR) patients and prostate cancer tissue using Quick-DNAâ„¢ FFPE Kit (Zymo Research) with and without pre-heating treatment in KOH/NOH solution. Primers for Androgen Receptor (AR) gene and four different PCR kits: MyTaq HS Red Mix 2X (BioLine), FastStart Taq DNA Polymerase (Roche), KAPA2G fast PCR Kit 2X (KAPA Biosystem) and KOD FX Neo (Toyobo) were used for amplification. DNA electrophoresis was performed to compare the PCR results. BioLine and Toyobo kits gave better PCR results than those of Roche and KAPA Biosystem. Increasing amount of Taq polymerase and dNTPs of Roche kit by two-fold could increase the quality of PCR results. Toyobo could amplify DNA up to 417 bp, however, none of these PCR kits could amplify DNA above 450 bp. Pre-heated treatment of FFPE tissue in NaOH/KOH did not improve the DNA quality and PCR results. Toyobo PCR ready-to-use kit gave the best result among the other three PCR kits used in this study in amplifying DNA isolated from FFPE tissue. Designing the primers producing amplicon not more than 450 bp is suggested.

Keywords

Amplification DNA-FFPE DNA Isolation PCR kit Tissue.

Zada, A., Susanto, P. S., Putri, R. L., Sahiratmadja, E. K., Hernowo, B. S., Rezano, A., & Sribudiani, Y. (2018). Optimization of DNA isolation method from Formalin-Fixed-Paraffin-Embedded tissues (FFPE) and comparative performance of four different Polymerase Chain Reaction (PCR) kits. Cellular and Molecular Biology, 64(13), 38–41. https://doi.org/10.14715/cmb/2018.64.13.8

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References

Dietrich D, Uhl B, Sailer V, Holmes EE, Jung M, Meller S et al. Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin Embedded Tissues by Overcoming PCR Inhibition. PLoS ONE, 2013, 8(10), e77771.
Bereczki L, Kis G, Bagdi E, Krenacs L. Optimization of PCR amplification for B- and T-cell clonality analysis on formalin-fixed and paraffin-embedded samples. Pathol Oncol Res 2007,13, 209-14.
Webster JD, Miller MA, DuSold D, Ramos-Vara J. Effects of prolonged formalin fixation on diagnostic immunohistochemistry in domestic animals. J Histochem Cytochem 2009, 57(8),753-61.
Kuykendall JR, Bogdanffy MS. Efficiency of DNA-histone crosslinking induced by saturated and unsaturated aldehydes in vitro. Mutat Res 1992,283(2), 131-6.
Bonin S, Petrera F, Niccolini B, Stanta G. PCR analysis in archival postmortem tissues. Mol Pathol 2003,156,184-6.
Shi SR, Datar R, Liu C, Wu L, Zhang Z, Cote RJ et al. DNA extraction from archival formalin-fixed, paraffin-embedded tissues: Heat-induced retrieval in alkaline solution. Histochem Cell Biol 2004,122(3),211-8.
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.
Zsikla V, Baumann M, Cathomas G. Effect of buffered formalin on amplification of DNA from paraffin wax embedded small biopsies using real-time PCR. J Clin Pathol 2004,57,654-6.
Gilbert MT, 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.
Shi SR, Cote RJ, Wu L, 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),1005-11
Bielawski K, Zaczek A, Lisowska U, et al (2001). The suitability of DNA extracted from formalin-fixed, paraffin-embedded tissues for double differential polymerase chain reaction analysis. Int J Mol Med 2001,8(5), 573-8.
Inadome Y, Noguchi M. Selection of higher molecular weight genomic DNA for molecular diagnosis from formalin-fixed material. Diagn Mol Pathol 2003, 12(4), 231-6.
http://www.toyobo-global.com/seihin/xr/lifescience/support/manual/KFX-201.pdf
https://www.bioline.com/mytaq-hs-red-mix.html
https://www.kapabiosystems.com/document/kapa2g-fast-hotstart-readymix-tds/?dl=1
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/.../Roche/.../fpcrnrobul.pdf

References

Dietrich D, Uhl B, Sailer V, Holmes EE, Jung M, Meller S et al. Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin Embedded Tissues by Overcoming PCR Inhibition. PLoS ONE, 2013, 8(10), e77771.

Bereczki L, Kis G, Bagdi E, Krenacs L. Optimization of PCR amplification for B- and T-cell clonality analysis on formalin-fixed and paraffin-embedded samples. Pathol Oncol Res 2007,13, 209-14.

Webster JD, Miller MA, DuSold D, Ramos-Vara J. Effects of prolonged formalin fixation on diagnostic immunohistochemistry in domestic animals. J Histochem Cytochem 2009, 57(8),753-61.

Kuykendall JR, Bogdanffy MS. Efficiency of DNA-histone crosslinking induced by saturated and unsaturated aldehydes in vitro. Mutat Res 1992,283(2), 131-6.

Bonin S, Petrera F, Niccolini B, Stanta G. PCR analysis in archival postmortem tissues. Mol Pathol 2003,156,184-6.

Shi SR, Datar R, Liu C, Wu L, Zhang Z, Cote RJ et al. DNA extraction from archival formalin-fixed, paraffin-embedded tissues: Heat-induced retrieval in alkaline solution. Histochem Cell Biol 2004,122(3),211-8.

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.

Zsikla V, Baumann M, Cathomas G. Effect of buffered formalin on amplification of DNA from paraffin wax embedded small biopsies using real-time PCR. J Clin Pathol 2004,57,654-6.

Gilbert MT, 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.

Shi SR, Cote RJ, Wu L, 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),1005-11

Bielawski K, Zaczek A, Lisowska U, et al (2001). The suitability of DNA extracted from formalin-fixed, paraffin-embedded tissues for double differential polymerase chain reaction analysis. Int J Mol Med 2001,8(5), 573-8.

Inadome Y, Noguchi M. Selection of higher molecular weight genomic DNA for molecular diagnosis from formalin-fixed material. Diagn Mol Pathol 2003, 12(4), 231-6.

http://www.toyobo-global.com/seihin/xr/lifescience/support/manual/KFX-201.pdf

https://www.bioline.com/mytaq-hs-red-mix.html

https://www.kapabiosystems.com/document/kapa2g-fast-hotstart-readymix-tds/?dl=1

https://www.sigmaaldrich.com/content/dam/sigma-aldrich/.../Roche/.../fpcrnrobul.pdf

Author Biography

Andri Rezano, Department of Biomedical Sciences, Division of Cell Biology, Faculty of Medicine, Universitas Padjadjaran, Bandung, West Java, Indonesia