DNA isolated from formalin-fixed paraffin-embedded healthy tissue after 30 years of storage can be used for forensic studies.
Author(s): Vitošević K, Todorović M, Slović Ž, Varljen T, Matić S, Todorović D
Publication: Forensic Sci Med Pathol, 2021, Vol. 17, Page 47-57
PubMed ID: 33159288 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of storing formalin-fixed paraffin-embedded (FFPE) blocks for up to 30 years and the methods of DNA extraction on the purity, yield, integrity and amplifiability of DNA from postmortem heart, liver and brain specimens. Additionally, the authors investigated the effects of storage on specimen histology.
Conclusion of Paper
The nuclear and cytological details, as well as the tissue architecture, were similar in sections H&E-stained immediately after embedding and those stained after 1-30 years of storage. The mean DNA purity and yield were higher when extraction was with phenol-chloroform than the PureLink Kit (1.92-2.05 versus 1.66-1.77, respectively, and 154.5-272.9 µg versus 105.9-104.4 µg, respectively). When extraction was with phenol-chloroform, the highest purity and yields were obtained from liver specimens, but yields were not dependent on tissue type when extraction was with the PureLink Kit. DNA yields were significantly lower from FFPE specimens stored for 2-6 years than those stored for <1 year (P<0.001), regardless of extraction method. PCR amplification success for the three amplicons declined with storage (r=-0.45 for phenol chloroform and r=-0.36 for the PureLink Kit) and was higher when extraction was with phenol chloroform than the PureLink Kit (P<0.001, all timepoints). The smallest amplicon (150 bp of glycerol-3-phosphate dehydrogenase 1, GPD1) was successfully amplified in 91%, 67%, 52%, 38%, 11%, 24% and 17% of specimens stored for <1 year, 2-6 years, 7-11 years, 12-16 years, 17-21 years, 22-26 years, and 27-30 years, respectively, when isolated with phenol-chloroform but only 67%, 3%, 4% and 0% of specimens stored for <1 year, 2-6 years, 7-11 years, and >12 years, respectively, when extraction was with the PureLink Kit. Similarly, the 262 bp fragments of β-Actin (ACTB) were successfully amplified in 91%, 36%, 22%, 4%, 0%, 7% and 0% of specimens stored for <1 year, 2-6 years, 7-11 years, 12-16 years, 17-21 years, 22-26 years, and 27-30 years, respectively, when isolated with phenol-chloroform but only 67%, 2%, 1% and 0% of specimens stored for <1 year, 2-6 years, 7-11 years, and >12 years, respectively, when extraction was with the PureLink Kit. Amplification success was even lower for the 407 bp fragment of ribosomal protein L4 (RPL4). The 407 bp fragment of RPL4 was only successfully amplified in 48% and 2% of specimens stored for <1 year and 2-6 years, respectively, when isolated with phenol-chloroform and was not amplifiable in any specimens stored for >7 years or after extraction with the PureLink Kit. When broken down by tissue, PCR success rates were higher for heart and brain specimens than liver (P<0.001, both) but were comparable in heart and brain.
Studies
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Study Purpose
This study investigated the effects of storing formalin-fixed paraffin-embedded (FFPE) blocks for up to 30 years and the methods of DNA extraction on the purity, yield, integrity and amplifiability of DNA from postmortem heart, liver and brain specimens. Additionally, the authors investigated the effects of storage on specimen histology. Additionally, the authors investigated the effects of FFPE block storage on specimen histology. In total, 181 heart, 181 liver and 181 brain specimens were collected 12 to 24 h after death at autopsy from 181 healthy patients who died suddenly (homicide, suicide or car accident). Specimens were formalin-fixed (details not provided), paraffin-embedded and were stored as paraffin blocks at room temperature for 1-30 years before the study. To investigate the effects of storage on histomorphology, tissue sections were stained with hematoxylin and eosin (H&E) immediately after paraffin embedding and again after storage. The tissue was digested overnight in Proteinase K at 56°C, and DNA was extracted using either phenol-chloroform-isoamyl alcohol or the PureLink Genomic DNA Kit. DNA was quantified using the spectrophotometer. DNA integrity was assessed by PCR amplification of a 150 bp fragment of GPD1, a 262 bp fragment of ACTB and a 407 bp fragment of RPL4 in conjunction with gel electrophoresis.
Summary of Findings:
The nuclear and cytological details, as well as the tissue architecture, were similar in sections H&E-stained immediately after embedding and those stained after 1-30 years of storage. The mean DNA purity and yield were higher when extraction was with phenol-chloroform than the PureLink Kit (1.92-2.05 versus 1.66-1.77, respectively, and 154.5-272.9 µg versus 105.9-104.4 µg, respectively). When extraction was with phenol-chloroform, the highest purity and yields were obtained from liver specimens, but yields were not dependent on tissue type when extraction was with the PureLink Kit. DNA yields were significantly lower from FFPE specimens stored for 2-6 years than those stored for <1 year (P<0.001), regardless of extraction method. PCR amplification success for the three amplicons declined with storage (r=-0.45 for phenol chloroform and r=-0.36 for the PureLink Kit) and was higher when extraction was with phenol chloroform than the PureLink Kit (P<0.001, all timepoints). The smallest amplicon (150 bp of glycerol-3-phosphate dehydrogenase 1, GPD1) was successfully amplified in 91%, 67%, 52%, 38%, 11%, 24% and 17% of specimens stored for <1 year, 2-6 years, 7-11 years, 12-16 years, 17-21 years, 22-26 years, and 27-30 years, respectively, when isolated with phenol-chloroform but only 67%, 3%, 4% and 0% of specimens stored for <1 year, 2-6 years, 7-11 years, and >12 years, respectively, when extraction was with the PureLink Kit. Similarly, the 262 bp fragments of β-Actin (ACTB) were successfully amplified in 91%, 36%, 22%, 4%, 0%, 7% and 0% of specimens stored for <1 year, 2-6 years, 7-11 years, 12-16 years, 17-21 years, 22-26 years, and 27-30 years, respectively, when isolated with phenol-chloroform but only 67%, 2%, 1% and 0% of specimens stored for <1 year, 2-6 years, 7-11 years, and >12 years, respectively, when extraction was with the PureLink Kit. Amplification success was even lower for the 407 bp fragment of ribosomal protein L4 (RPL4). The 407 bp fragment of RPL4 was only successfully amplified in 48% and 2% of specimens stored for <1 year and 2-6 years, respectively, when isolated with phenol-chloroform and was not amplifiable in any specimens stored for >7 years or after extraction with the PureLink Kit. When broken down by tissue, PCR success rates were higher for heart and brain specimens than liver (P<0.001, both) but were comparable in heart and brain.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Autopsy
Platform:
Analyte Technology Platform DNA Spectrophotometry Morphology H-and-E microscopy DNA PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Biospecimen location Heart
Brain
Liver
PCR Specific Length of gene fragment 407 bp fragment of RPL4
262 bp fragments of ACTB
150 bp of GPD1
Analyte Extraction and Purification Analyte isolation method PureLink Kit
Phenol-chloroform-isoamyl alcohol
Storage Storage duration <1 year
2-6 years
7-11 years
12-16 years
17-21 years
22-26 years
27-30 years