4976 - Optimization of automated germline DNA extraction from non-tumoral formalin-fixed paraffin embedded (FFPE) tissues

Background

Recent studies have identified a complex but definitive role of germline DNA in cancer predisposition. Archived formalin-fixed paraffin-embedded (FFPE) samples from cancer patients are vital for several molecular and genomic studies. For retrospective studies investigating gene mutations, loss of heterozygosity and copy number changes, non-tumoral FFPE samples can be used as a source of germline DNA. The key challenge with FFPE DNA purification is usually due to the fixation process, which causes cross-linking and fragmentation of FFPE DNA. In spite of the development of several techniques for FFPE DNA extraction, automated extraction can provide efficient DNA purification with the least hands-on and the least contamination. We compared two different automated approaches with special focus on DNA yield and quality using the DNA integrity number (DIN) value.

Methods

The study was carried out on 48 non-tumoral FFPE samples from cancer patients. Two FFPE pretreatment methods were used simultaneously: GeneRead DNA FFPE Kit (removes cytosine deamination artifacts from FFPE) and QIAsymphony DSP DNA Kit. After the initial pretreatment, the extraction step was performed using the automated QIAsymphony SP instrument for both methods. Finally, the purified DNA was assessed using TapeStation for measuring the concentration and DIN value.

Results

The median DNA concentration using the GeneRead method was 13.85 ng/ul (1.13-111 ng/ul), while for QIAsymphony DSP the median DNA was 5.3 ng/ul (1.07-156 ng/ul). Of the total 48 FFPE samples, 40 purified by GeneRead and 29 purified by QIAsymphony DSP have DNA concentrations above the functional quantitative range of DIN. The median DIN value was 3.3 and 4.1 for GeneRead and QIAsymphony DSP, respectively.

Conclusions

This study demonstrates that the FFPE pretreatment step has an effect on automated DNA extraction. Highly efficient extraction of FFPE DNA based solely on quantity can be misleading as the DNA may be highly fragmented. The removal of cytosine deamination artifacts from FFPE samples can enhance DNA purification with less degradation. This may also have a crucial influence on the downstream molecular approaches such as DNA sequencing.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The author.

Funding

Has not received any funding.

Disclosure

The author has declared no conflicts of interest.