Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Improved protocol for extraction of genomic DNA from formalin-fixed paraffin-embedded tissue samples without the use of xylene.

Author(s): Kikuchi A, Sawamura T, Daimaru O, Horie M, Sasaki K, Okita N

Publication: Clin Chem Lab Med, 2016, Vol. 54, Page e375-e377

PubMed ID: 27171389 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of deparaffinization using one of four solvents in a mixing-based method rather than a standard xylene-based method on the yield, purity, and integrity of DNA isolated from FFPE gastric tumor specimens. Surgical resection specimens from 29 patients with gastric cancer, for which details of specimen processing including fixation and embedding methods were not provided, were serially sectioned. Two 5 µm sections of each block were deparaffinized by incubation in xylene for 5 min followed by centrifugation and washing in ethanol before resuspension in lysis buffer. Similarly, two 5µm sections of each block were deparaffinized with each of hexadecane, pentadecane, tetradecane, and mineral oil by placing the sections in the desired solvent, vortexing for 10 s, incubation at 56˚C for 5 min, addition of lysis buffer to the tube containing the solvent, and centrifugation before lysis. For all deparaffinization methods, specimens were Proteinase K digested at 56˚C for 60 min and then demodified by heating to 90˚C for 60 min, but this was done in the tube with the solvent above the lysate (mix method) for all solvents other than xylene. DNA was extracted from the lysates using the HighPure FFPET DNA Isolation kit, quantified fluorometrically using the Qubit dsDNA BR Assay Kit, and the purity determined spectrophotometrically. DNA integrity was assessed by amplification of different fragment lengths of the GAPDH gene.

   Summary of Findings:

   Although the purity and cycle threshold values for the three GAPDH products were comparable among all depraffinization methods, DNA yields were on average 1.18 to 1.41-fold higher when specimens were deparaffinized using a non-xylene solvent and the mix method. The highest average yields were obtained when deparaffinized with hexadecane (57.3 ng/µL), followed by mineral oil (52.3 ng/µL), tetradecane (49.1 ng/µL), and pentadecane (48.2 ng/µL).

   Biospecimens

   • Tissue - Stomach

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	PCR
   DNA	Fluorometry
   DNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Deparaffinization	Hexadecane Xylene Pentadecane Tetradecane Mineral oil
   PCR Specific	Length of gene fragment	60 bp 100 bp 200 bp
   PCR Specific	Targeted nucleic acid	GAPDH

   View more