Nucleic acids from long-term preserved FFPE tissues are suitable for downstream analyses.

Author(s): Ludyga N, Grünwald B, Azimzadeh O, Englert S, Höfler H, Tapio S, Aubele M

Publication: Virchows Arch, 2012, Vol. 460, Page 131-40

PubMed ID: 22270699 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of formalin fixation, storage of FFPE blocks, DNA and RNA extraction method and lysis duration on the quantity, purity and quality of DNA and RNA from 6 breast cancer specimens and 1 colon cancer specimen. The FFPE colon cancer specimen had a matched frozen specimen. The breast cancer specimens were stored for 10-40 years before extraction.

   Summary of Findings:

   DNA yield was higher when DNA was extracted from FFPE tissues using phenol-chloroform than either of the commercial kits, but RNA yield was highest using the QIAamp kit followed by the Norgen FFPE kit. The DNA purity was highest following phenol-chloroform or QIAamp extraction, but 2 of 7 QIAamp purified specimens were slightly contaminated with protein. The RNA was generally free of protein contamination, but when purified with Norgen FFPE, it was contaminated with solvents, salts and organic compounds. RINs and DNA fragmentation were not affected by extraction method, but RINs were much higher for RNA extracted from frozen colon specimens than FFPE colon specimens (8.7 versus 2.20). The longest PCR fragments were amplified using DNA extracted using phenol-chloroform. A 1 day lysis step, in conjunction with phenol-chloroform or Norgen FFPE kit extraction, allowed for amplification of fragments that were not amplifiable after a 1 h lysis step and increased intensity of the bands generated using QIAamp extracted DNA. Interestingly, DNA and RNA yields were lower from specimens stored 10-12 years than specimens stored 16-40 years, but the DNA was much more fragmented when FFPE specimens were stored for 16-40 years rather than 10-12 years.

   Biospecimens

   • Tissue - Breast
   • Tissue - Colorectal

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Electrophoresis
   DNA	Spectrophotometry
   DNA	PCR
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	10-40 years
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   Analyte Extraction and Purification	Analyte isolation method	QIAamp DNA FFPE kit Norgen FFPE RNA/DNA purification kit Phenol-chloroform
   Analyte Extraction and Purification	Protein digestion	1 h 1 day

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