Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Towards molecular autopsies: Development of a FFPE tissue DNA extraction workflow.

Author(s): Viljoen R, Reid KM, Mole CG, Rangwaga M, Heathfield LJ

Publication: Sci Justice, 2022, Vol. 62, Page 137-144

PubMed ID: 35277226 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the impact of deparaffinization method, section thickness, section number, extraction method, and block storage duration on the yield, integrity, and purity of DNA isolated from postmortem heart and lung tissues. FFPE blocks containing heart and lung tissues from patients (2-6 cases per experiment)-that experienced a sudden unexpected death were stored for 10 months-3.5 years prior to use. No other details of specimen acquisition and processing were provided. To test the effects of deparaffinization method, one or two 10 µm-thick sections of heart and lung blocks from two cases were placed in a tube and deparrafinized with (i) xylene (1 h at room temperature) followed by rehydration through a graded ethanol series, (ii) mineral oil (1 h at 90°C), or (iii) Zymo research deparaffinization solution (1 min at 55°C) before extraction with the FFPE DNA MiniPrepTM kit. To explore effects of extraction method, FFPE sections deparaffinized with Zymo research deparaffinization solution or mineral oil were used for DNA extraction with the (i) ZR FFPE DNA MiniPrepTM, (ii) Nucleospin DNA FFPE XS, and (iii) QIAamp DNA FFPE Tissue Kits. To evaluate effects of tissue type and the number of sections used for DNA extraction, DNA was extracted from 5 to 100 FFPE sections that were 1, 3, or 5 um thick from three patients with the QIAamp FNA FFPE Tissue Kit with a 20 h lysis step following deparaffinization with the Zymo research deparaffinization solution. To investigate the effect of FFPE block storage duration, DNA was extracted from 30, 50, and 100 1 µm-thick sections of FFPE blocks stored 2.5-3.5 years using QIAamp DNA FFPE tissue kit. DNA was quantified using the real-time PCR-based Quantifiler Trio DNA quantification Kit and the Qubit dsDNA HS Assay Kit. DNA purity was evaluated using NanoDrop Spectrophotometry and DNA integrity was assessed by Genomic DNA ScreenTape on the 4200 TapeStation System. To ensure there was no contamination, DNA was profiled using the Promega PowerPlex ESX 16 System.

   Summary of Findings:

   Median DNA yield was higher when two 10 µm-thick FFPE sections were used for DNA extraction than when one 10 µm-thick section was used (P=0.0006). DNA yields were comparable among the three deparaffinization methods. From one case, heart tissue yielded more DNA than lung, but from the other case lung yielded more DNA; microscopy review showed this was attributable to the integrity of the tissue. DNA yields from heart and lung were highest when extraction was with the QIAamp kit, regardless of whether deparaffinization was with Zymo deparaffinization solution or mineral oil. Further of the three DNA extraction methods examined, only the QIAamp kit yielded DNA with a high level of purity (based on a ratio of absorbance at 260 to 280 nm that was >1.8). Mean DNA yield was 8.8- and 26.8-fold higher when 30-100 1 µm thick sections were used for extraction compared to when 5-10 3 µm thick (nonsignificant, P>0.05) or 5 µm thick sections were used (P<0.05).  While mean DNA integrity number (DIN) was significantly lower when DNA was extracted from 30-100 1 µm-thick sections than when 5-10 3 µm- thick or 5 µm-thick sections were used (P<0.05 and P<0.005, respectively), extracts from 1 µm-thick sections had a larger percentage of DNA fragments >400 bp than when 3 µm-thick or 5 µm-thick sections were used (55% versus 22% and 0%, respectively). DNA purity, assessed by the ratio of absorbance at 260 to 230 or 280, was higher when sections used for extraction were 1 µm-thick versus 3 µm-thick (P=0.0093 and P=0.0005, respectively) or 5 µm-thick (P=0.0001 and P<0.0001, respectively). Increasing the number of 1 µm-thick sections used for extraction resulted in increases in DNA yield, without affecting purity or integrity. DNA yield was not correlated with FFPE block storage duration, but there was a modest negative correlation between DNA purity (A260/230 ratio) and storage duration (r=-0.476, P=0.046). DNA profiling revealed that each extract only contained DNA from a single source.

   Biospecimens

   • Tissue - Lung
   • Tissue - Heart

   Preservative Types

   • Formalin

   Diagnoses:

   • Autopsy

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Biospecimen location	Heart tissue Lung tissue
   Biospecimen Aliquots and Components	Aliquot size/volume	1 section 2 sections 30 sections 50 sections 100 sections 3 µm-thick FFPE sections (5-10) 5 µm-thick FFPE sections (5-10) 1 µm-thick FFPE sections (30-100)
   Storage	Storage duration	450-1320 days
   Analyte Extraction and Purification	Analyte isolation method	Nucleospin DNA FFPE XS kit QIAamp DNA FFPE Tissue Kit ZR FFPE DNA MiniPrepTM Kit
   Analyte Extraction and Purification	Deparaffinization	Xylene (1 h at room temperature) followed by graded ethanol series Mineral oil (1 h at 90°C) Zymo research deparaffinization solution (1 min at 55°C)

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