Evaluation and Comparison of Genomic DNA Extraction Methods and PCR Optimization on Archival Formalin-Fixed and Paraffin-Embedded Tissues of Oral Squamous Cell Carcinoma.
Author(s): Khan SS, Tijare M, Kasetty S, Jain M, Alamoudi A, Bahammam HA, Bahammam SA, Bahammam MA, Varadarajan S, Raj AT, Patil S
Publication: Diagnostics (Basel), 2022, Vol. 12, Page
PubMed ID: 35626372 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare DNA yield, purity and p53 amplification in DNA extracted from formalin-fixed paraffin-embedded (FFPE) sections that were deparaffinized using either xylene or heat, as well as in DNA extracted from oral squamous cell carcinoma (OSCC) specimens that were stored in formalin long-term prior to DNA isolation with phenol-chloroform or the HiPurATM Paraffin-Embedded Tissue DNA Purification Spin Kit.
Conclusion of Paper
The mean DNA yield, the percentage of specimens with acceptable purity (a ratio of absorbance at 260/280 nm between 1.6-1.8) and the percentage of specimens for which p53 amplification was successful were all highest when FFPE sections were deparaffinized in xylene and DNA was extracted using phenol-chloroform, followed by specimens extracted with phenol-chloroform after heat deparaffinization. For both FFPE and long-term formalin-fixed specimens, DNA yields were higher when extraction was with phenol-chloroform rather than the HiPurATM Paraffin-Embedded Tissue DNA Purification Spin Kit. The percentage of specimens with acceptable purity and successful p53 amplification was lower for long-term formalin-stored specimens than FFPE specimens.
Studies
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Study Purpose
The purpose of this study was to compare DNA yield, purity and p53 amplification in DNA extracted from xylene or heat deparaffinized FFPE sections, as well as in DNA extracted from OSCC specimens that were stored long-term in formalin prior to extraction with phenol-chloroform or the HiPurATM Paraffin-Embedded Tissue DNA Purification Spin Kit. FFPE (45) and long-term formalin-fixed specimens (30) were selected from the archive. FFPE specimens were collected between 2003-2011 and fixed in formalin for 24-48 h before paraffin embedding, but no further details were provided. FFPE blocks were assigned to one of three extraction procedures: (i) deparaffinization in 2-3 changes of xylene (60°C for 30 min) and extraction with phenol-chloroform (unspecified number of 10 µm sections of 15 blocks), (ii) deparaffinization by heating slide-mounted FFPE sections on a hot plate (temperature not specified), wax removal by blotting, and extraction with phenol-chloroform (unspecified number of 10 µm sections of 15 blocks) or (iii) deparaffinization in 3-4 changes of room temperature xylene (5 min each) and DNA extraction with the HiPurATM Paraffin-Embedded Tissue DNA Purification Spin Kit. Specimens that were stored long-term in formalin were collected between 2003-2011 but no further details were provided. These formalin-fixed specimens were frozen in liquid nitrogen and pulverized with a mortar and pestle. DNA was extracted from the powder using phenol-chloroform or the HiPurATM Paraffin-Embedded Tissue DNA Purification Spin Kit. DNA yield and purity were assessed using a Picodrop UV-Spectrophotometer. DNA integrity was evaluated qualitatively by gel electrophoresis of p53 PCR amplification products.
Summary of Findings:
The mean DNA yield was higher when FFPE sections were deparaffinized in xylene and DNA was extracted using phenol-chloroform than when extracted with phenol-chloroform after heat deparaffinization (129.64 ng/µL versus 50.04 ng/µL, P<0.05 both) or extracted using the HiPurATM kit after xylene deparaffinization (129.64 ng/µL versus 36.43 ng/µL, P<0.01). The percentage of specimens with a ratio of absorbance at 260/280 between 1.6-1.8 (acceptable purity) was lower in specimens extracted using the HiPurATM Kit after xylene deparaffinization than when deparaffinized in xylene and DNA was extracted using phenol-chloroform (66.67% versus 26.67%). The mean DNA yield from specimens stored long-term in formalin was higher when DNA was extracted using phenol-chloroform rather than the HiPurATM Paraffin-Embedded Tissue DNA Purification Spin Kit (31.94 versus 7.526 ng/µL, P<0.001), but the percentage of specimens with acceptable purity was comparable (26.67% and 13.33%, respectively). The percentage of specimens that yielded extracted DNA with acceptable purity was higher from xylene deparaffinized FFPE sections than specimens stored long-term in formalin that were extracted with phenol-chloroform (66.67% versus 26.67%, P=0.02), but the difference was not significant when the FFPE specimens were heat deparaffinized. Further, while the percentage of specimens with extracted DNA of acceptable purity was slightly higher from FFPE specimens than specimens stored long-term in formalin when extraction was with the HiPurATM Paraffin-Embedded Tissue DNA Purification Spin Kit, the difference was not significant (26.67% versus 13.33%, P=0.37). Following the pattern observed for yield and purity, the percentage of specimens with successful p53 amplification was highest when DNA was extracted from FFPE sections using phenol-chloroform after xylene deparaffinization (93.33%), followed by DNA extracted from FFPE sections using phenol-chloroform after heat deparaffinization (86.66%), and DNA extracted from FFPE sections using the HiPurATM Paraffin-Embedded Tissue DNA Purification Spin Kit (66.66%), and lowest when DNA was extracted from specimens stored long-term in formalin with phenol-chloroform (46.66%) or the HiPurATM Paraffin-Embedded Tissue DNA Purification Spin Kit (53.33%).
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Spectrophotometry DNA PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method Phenol-chloroform
HiPurATM Paraffin-Embedded Tissue DNA Purification Spin Kit
Biospecimen Preservation Embedding medium Paraffin
Not embedded
Biospecimen Preservation Time in fixative 24-48 h
Years
Analyte Extraction and Purification Deparaffinization Xylene
Heat