Research Article Open Access

An Effective and Viable DNA Extraction Protocol for FFPE Tissues and its Effect on Downstream Molecular Application

Sai Sushmitha Kontham1, Charles Emmanuel Jebaraj Walter1, Manjula Raju1, Chinambedu Dandapani Mohanapriya1, Nirmala Karuppasamy1, Zioni Sangeetha Shankaran1 and Thanka Johnson1
  • 1 Sri Ramachandra Institute of Higher Education and Research, India

Abstract

Archival Formalin-Fixed Paraffin-Embedded (FFPE) tissues are a readily available source of DNA for retrospective studies including genetic predisposition. FFPE tissues present significant challenges for extraction of pure genomic DNA in adequate amounts due to the harsh fixation conditions and long-term storage associated with samples. The aim of the study is to propose a simple, in-expensive, viable protocol for the extraction of DNA from FFPE tissues and to optimize the same for the detection of 3’UTR miRNA binding site polymorphism in ADAMTS1 gene in colorectal cancer tissues and normal colonic mucosa archival tissues using Sanger sequencing and TaqMan SNP genotyping assay. Five different DNA extraction methods were compared and analyzed. NanoDrop quantification demonstrated that out of the various methods tested, highest quantity and pure DNA was obtained from Modified non heating extraction. When we further applied this method for colorectal cancer tissues and normal colonic mucosa archival tissues the NanoDrop readout indicated that the DNA was intact and pure. Intra samples comparison over a time period of 6 months revealed that the DNA was intact. The samples isolated with Modified non heating extraction were found suitable for downstream processes such as Sanger sequencing and TaqMan SNP genotyping assay. This cost effective and non-arduous protocol can successfully extract even minute quantities of DNA from FFPE tissues and facilitate the downstream molecular analysis of a large number of archival specimens for retrospective studies.

American Journal of Biochemistry and Biotechnology
Volume 16 No. 1, 2020, 148-161

DOI: https://doi.org/10.3844/ajbbsp.2020.148.161

Submitted On: 10 January 2020 Published On: 24 March 2020

How to Cite: Kontham, S. S., Walter, C. E. J., Raju, M., Mohanapriya, C. D., Karuppasamy, N., Shankaran, Z. S. & Johnson, T. (2020). An Effective and Viable DNA Extraction Protocol for FFPE Tissues and its Effect on Downstream Molecular Application. American Journal of Biochemistry and Biotechnology, 16(1), 148-161. https://doi.org/10.3844/ajbbsp.2020.148.161

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Keywords

  • FFPE Tissues
  • Sanger Sequencing
  • Colorectal Cancer
  • Modified Non Heating Extraction
  • TaqMan SNP Genotyping Assay