A simple procedure for the extraction of DNA from long-term formalin-preserved brain tissues for the detection of EBV by PCR

Asma Hassani, Gulfaraz Khan

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

Long-term formalin fixed brain tissues are potentially an important source of material for molecular studies. Ironically, very few protocols have been published describing DNA extraction from such material for use in PCR analysis. In our attempt to investigate the role of Epstein-Barr virus (EBV) in the pathogenesis of multiple sclerosis (MS), extracting PCR quality DNA from brain samples fixed in formalin for 2-22. years, proved to be very difficult and challenging. As expected, DNA extracted from these samples was not only of poor quality and quantity, but more importantly, it was frequently found to be non-amplifiable due to the presence of PCR inhibitors. Here, we describe a simple and reproducible procedure for extracting DNA using a modified proteinase K and phenol-chloroform methodology. Central to this protocol is the thorough pre-digestion washing of the tissues in PBS, extensive digestion with proteinase K in low SDS containing buffer, and using low NaCl concentration during DNA precipitation. The optimized protocol was used in extracting DNA from meninges of 26 MS and 6 non-MS cases. Although the quality of DNA from these samples was generally poor, small size amplicons (100-200 nucleotides) of the house-keeping gene, β-globin could be reliably amplified from all the cases. PCR for EBV revealed positivity in 35% (9/26) MS cases, but 0/6 non-MS cases. These findings indicate that the method described here is suitable for PCR detection of viral sequences in long-term formalin persevered brain tissues. Our findings also support a possible role for EBV in the pathogenesis of MS.

Original languageEnglish
Pages (from-to)558-563
Number of pages6
JournalExperimental and Molecular Pathology
Volume99
Issue number3
DOIs
Publication statusPublished - Dec 1 2015

Keywords

  • DNA extraction
  • Epstein-Barr virus
  • Formalin preserved tissue
  • Multiple sclerosis

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Biology
  • Clinical Biochemistry

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