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

Viljoen, Rabia; Reid, Kate Megan; Mole, Calvin Gerald; Rangwaga, Mmannini; Heathfield, Laura Jane

doi:10.1016/j.scijus.2021.12.005

Cited by 2 publications

(2 citation statements)

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“…Frazer et al previously reported that neither the ΔCq of the Illumina QC assay nor DIN was changed between 24 and 72 h of proteinase K digestion [28]. Viljoen et al compared tissue thicknesses of 1 μm/3 μm/5 μm and reported that the thinner the tissue section is, the higher is the DNA concentration and purity, and the lower is the DIN [20]. Our results overlap with these reports.…”

Section: Discussionsupporting

confidence: 73%

“…DNA in postmortem FFPE tissues is generally known to be poorly preserved [16][17][18] and, therefore, not applicable for use in further molecular biology research. Some studies have discussed DNA recovery from FFPE tissues obtained from medicolegal autopsies, but the tissue sampling method for such autopsies is not the same as that for clinical autopsies [19,20]. Very few reports have evaluated the DNA quality of clinical autopsy FFPE tissues: Funabashi et al compared DNA extraction methods for archived FFPE samples, but they only selected "well-preserved" samples [21].…”

Section: Introductionmentioning

confidence: 99%

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Optimal DNA quality preservation process for comprehensive genomic testing of pediatric clinical autopsy formalin-fixed, paraffin-embedded tissues

Watanabe

Umezu

Kyushiki

et al. 2022

pjp

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Clinical autopsies are performed to reveal the process of the disease that caused patient death and validate the diagnosis and treatment decisions. In pediatric clinical autopsy, the feedback provided to bereaved families has a considerable social impact; however, pediatric diseases are diverse, which makes it difficult to elucidate them. Therefore, it is necessary to employ molecular biology techniques in addition to conventional methods. Formalin-fixed, paraffin-embedded (FFPE) tissues are routinely prepared. However, clinical autopsy FFPE tissue processing is not standardized, and it is unclear whether DNA from such tissues can be used for comprehensive genomic analysis. In this study, we evaluated the DNA quality of FFPE tissues from 15 recent autopsy cases at a single-center children's hospital using quantitative polymerase chain reaction [PCR (Q129/Q41)] and nanoelectrophoresis (DNA integrity number (DIN)). Good quality DNA was obtained from every organ type excluding bone marrow within 6 days of formalin fixation. Prolonged proteinase K digestion (48 h > 24 h > 1 h) and thicker tissue sections (10 μm > 1 μm) improved Q129/Q41; however, 24 h fixed FFPE tissues showed better DNA quality. We propose an optimal and feasible workflow for storing short-term fixed FFPE tissues as DNA-preserved FFPE tissues for future comprehensive genomic searches.

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Section: Discussionsupporting

confidence: 73%