Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation

PloS One
Naoki EinagaMariko Esumi

Abstract

Formalin-fixed, paraffin-embedded (FFPE) tissues used for pathological diagnosis are valuable for studying cancer genomics. In particular, laser-capture microdissection of target cells determined by histopathology combined with FFPE tissue section immunohistochemistry (IHC) enables precise analysis by next-generation sequencing (NGS) of the genetic events occurring in cancer. The result is a new strategy for a pathological tool for cancer diagnosis: 'microgenomics'. To more conveniently and precisely perform microgenomics, we revealed by systematic analysis the following three details regarding FFPE DNA compared with paired frozen tissue DNA. 1) The best quality of FFPE DNA is obtained by tissue fixation with 10% neutral buffered formalin for 1 day and heat treatment of tissue lysates at 95°C for 30 minutes. 2) IHC staining of FFPE tissues decreases the quantity and quality of FFPE DNA to one-fourth, and antigen retrieval (at 120°C for 15 minutes, pH 6.0) is the major reason for this decrease. 3) FFPE DNA prepared as described herein is sufficient for NGS. For non-mutated tissue specimens, no artifactual mutation occurs during FFPE preparation, as shown by precise comparison of NGS of FFPE DNA and paired frozen tissue DNA follo...Continue Reading

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Methods Mentioned

BETA
laser-capture microdissection
PCR
surgical resection
electrophoresis
amplicon sequencing
chip
deamination
MDA
amplicon

Software Mentioned

Ion Reporter
Ion Torrent Suite
IonXpress

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