ArticleBiomolecular detection and quantification2018
Using ddPCR to assess the DNA yield of FFPE samples.
Article in Biomolecular detection and quantification, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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Who cites it
8 citing papers in PubMed.
- Profiling of the tumor-associated microbiome in patients with hepatocellular carcinoma.Gut pathogens · 2025Article
- Digital PCR-Based Method for Detecting CDKN2A Loss in Brain Tumours.Molecular diagnosis & therapy · 2022Article
- A protocol for good quality genomic DNA isolation from formalin-fixed paraffin-embedded tissues without using commercial kits.Molecular biology reports · 2022Article
- Comparison of Droplet Digital PCR and Metagenomic Next-Generation Sequencing Methods for the Detection of Human Herpesvirus 6B Infection Using Cell-Free DNA from Patients Receiving CAR-T and Hematopoietic Stem Cell Transplantation.Infection and drug resistance · 2022Article
- High Clinical Impact of Broad-Range Fungal PCR in Suspected Fungal Sinusitis.Journal of clinical microbiology · 2021Article
- Impact of DNA integrity on the success rate of tissue-based next-generation sequencing: Lessons from nationwide cancer genome screening project SCRUM-Japan GI-SCREEN.Pathology international · 2020Article
- Sensitive Identification of Bacterial DNA in Clinical Specimens by Broad-Range 16S rRNA Gene Enrichment.Journal of clinical microbiology · 2020Article
- Improved Species-Level Clinical Identification of Enterobacteriaceae through Broad-RangeJournal of clinical microbiology · 2019Article
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5 authors.
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No grant is acknowledged in the PubMed record.
Abstract
objectivesDetection 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.
methodsWe 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.
resultsDeparaffinization 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.
conclusionsSamples 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.
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