Using ddPCR to assess the DNA yield of FFPE samples

X J David Lu; Kelly Y P Liu; Yuqi Sarah Zhu; Cindy Cui; Catherine F Poh

doi:10.1016/j.bdq.2018.10.001

Using ddPCR to assess the DNA yield of FFPE samples

Biomol Detect Quantif. 2018 Nov 7:16:5-11. doi: 10.1016/j.bdq.2018.10.001. eCollection 2018 Dec.

Authors

X J David Lu^{1

2}, Kelly Y P Liu^{1

2}, Yuqi Sarah Zhu², Cindy Cui², Catherine F Poh^{1

2

3}

Affiliations

¹ Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.
² Department of Integrative Oncology, British Columbia Cancer Research Centre, Vancouver, BC, V5Z 1L3, Canada.
³ Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, V6T 2B5, Canada.

Abstract

Objectives: Detection of genomic alterations in diseases can be achieved with current molecular technologies. However, the molecules extracted from formalin-fixed, paraffin-embedded (FFPE) bio-samples are often limited possibly due to DNA fragmentation and crosslinking caused by the sample fixation and processing. The study objective was to design a droplet digital PCR (ddPCR) assay to assess the quality and quantity of DNA derived from various DNA extraction conditions on FFPE samples.

Methods: We used 10 μm-thick sections from 5 FFPE oral tumoral blocks, each consisting of 10-15 sections. The protocol variables tested included: 1) tissue staining; 2) duration and 3) temperature of post-digestion heat treatment; and 4) DNA extraction method. DNA quantity was assessed using the NanoDrop 2000 (Thermo Fisher Scientific, USA), the Qubit fluorometer (Thermo Fisher Scientific, USA), and a ddPCR-based assay. DNA quality was assessed using a ddPCR assay for the degree of fragmentation and the effectiveness of removing crosslinks with varying guanine-cytosine (GC)-content.

Results: Deparaffinization with xylene helped to increase the DNA yield. Tissue staining (methyl green staining, pH 6) prior to microdissection, comparing to no staining, caused additional DNA fragmentation. Compared to column-based method, DNA extracted with phenol chloroform and ethanol precipitation increased the degree of fragmentation and lowered the yield of amplifiable DNA. The cross-linking derived from GC-contents may not be the only factor impacting on the DNA quality.

Conclusions: Samples undergoing different pre-treatment conditions prior to extraction can impact the yield of amplifiable DNA. Our ddPCR assay can be used to assess for both DNA quantity and quality.

Keywords: DNA extraction; DNA fragmentation; Droplet digital PCR; Formalin-fixed paraffin-embedded tissues; Formalin-induced crosslinking; GC-content.