Evaluation of DNA and RNA quality from archival formalin-fixed paraffin-embedded tissue for next-generation sequencing - Retrospective study in Japanese single institution.
Author(s): Fujii T, Uchiyama T, Matsuoka M, Myojin T, Sugimoto S, Nitta Y, Okabe F, Sugimoto A, Sekita-Hatakeyama Y, Morita K, Itami H, Hatakeyama K, Ohbayashi C
Publication: Pathol Int, 2020, Vol. 70, Page 602-611
PubMed ID: 32542983 PubMed Review Paper? No
Purpose of Paper
This paper assessed the quality of DNA and RNA isolated from formalin-fixed paraffin-embedded (FFPE) blocks stored for one to four years prior to extraction, as well as the suitability of DNA for next generation sequencing, in surgically resected tissue from nine different tumor types.
Conclusion of Paper
DNA and RNA extracted from FFPE sections displayed clear evidence of fragmentation compared to electrophoretic traces of DNA and RNA isolated from fresh cells (T24 UC cell line) and frozen tissue sections. 28S and 18S rRNA bands were also clearly visible in RNA isolated from the fresh cells and the frozen tissue sections but were not observable in RNA extracted from FFPE tissue sections.
DNA from FFPE tissue blocks stored for 1 year had significantly higher DNA integrity numbers (DINs) (p<0.01 for all), larger peak DNA fragment size (p<0.05 for all), and lower cycle threshold (Ct) values for EGFR (p<0.001 for all) compared to DNA from FFPE tissue blocks stored for longer durations (2-4 years). RNA from FFPE tissue blocks stored for 1 year also had significantly higher RNA integrity numbers (RINs) than RNA from blocks stored for longer durations (2-4 years, p<0.05 for all). Of the 108 FFPE tumor specimens assessed, 21 specimens yielded RNA with a DV200 value <30%, leading the authors to conclude that they were of insufficient quality for NGS library construction (not performed). DNA from twelve FFPE specimens including that with the highest DIN (1 y of FFPE block storage) and the lowest DIN (4 y of FFPE storage) were successfully analyzed by NGS.
Studies
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Study Purpose
This paper assessed the quality of DNA and RNA isolated from formalin-fixed paraffin-embedded (FFPE) blocks stored for one to four years prior to extraction, as well as the suitability of DNA for next generation sequencing, in surgically resected tissue from nine different tumor types. In total 108 surgically resected tissue specimens were assessed that included glioblastoma, squamous cell carcinoma (tongue), papillary throid carcinoma, lung adenocarcinoma, gastric adenocarcinoma, pancreatic adenocarcinoma, renal cell carcinoma, urothelial carcinoma, and endometrial carcinoma. Tumor specimens were procured over a period of four years, fixed in 10% neutral buffered formalin for 12-72 h, paraffin embedded, and stored as paraffin blocks for up to four years under unspecified conditions. Unstained sections (8 µm) were used for DNA extraction with the QIAamp DNA FFPE Tissue Kit following deparaffinization and proteinase K treatment. RNA was extracted using the miRNeasy FFPE Tissue Kit. DNA and RNA concentrations were quantified by spectrophotometry. DNA and RNA integrity were assessed by DNA integrity number (DIN) and RNA integrity number (RIN) using a TapeStation (value range: 1 (degraded) to 10 (intact)). DNA integrity was also assessed by qPCR amplification of EGFR (371 bp amplicon), and RNA integrity was also assessed by DV200. DNA isolated from twelve FFPE specimens were analyzed by targeted capture-based NGS using the Illumina MiniSeq sequencing platform after library construction with the AmpliSeq for Illumina Library Plus kit.
Summary of Findings:
DNA and RNA extracted from FFPE sections displayed clear evidence of fragmentation compared to electrophoretic traces of DNA and RNA from fresh cells (T24 UC cell line) and frozen tissue sections. 28S and 18S rRNA bands were also clearly visible in the RNA isolated fresh cells and the frozen tissue sections examined but were not observable in RNA extracted from FFPE tissue sections.
FFPE tissue blocks stored for 1 year yielded DNA with significantly higher DIN (p<0.01 for all), larger peak DNA fragment size (p<0.05 for all), and lower cycle threshold (Ct) values for EGFR (p<0.001 for all) compared to DNA from FFPE tissue blocks stored for longer durations (2-4 years). FFPE tissue blocks stored for 1 year also yielded RNA with significantly higher RINs than RNA from blocks stored for longer durations (2-4 years, p<0.05 for all). Of the 108 FFPE tumor specimens assessed, 21 specimens had RNA with DV200 value <30%, indicating that they were of insufficient quality for NGS library construction. DNA from twelve FFPE specimens including specimens with the highest DIN (1 y of FFPE block storage) and the lowest DIN (4 y of FFPE storage) were analyzed by NGS. Bands of the targeted amplicon size (294-304 bp) were detected in all libraries. The percentage of aligned reads were >90% for all specimens and the uniformity of coverage ranged between 86 and 100%, with no clear effect of storage duration. Somatic mutations with a frequency of 5% were detectable at a coverage of 500x in most cases.
Biospecimens
- Tissue - Brain
- Tissue - Thyroid Gland
- Tissue - Tongue
- Tissue - Lung
- Tissue - Endometrium
- Tissue - Pancreas
- Tissue - Kidney
- Tissue - Bladder
- Tissue - Stomach
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA Spectrophotometry DNA Automated electrophoresis/Bioanalyzer RNA Spectrophotometry RNA Real-time qRT-PCR DNA Next generation sequencing RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 1 y
2 y
3 y
4 y