HPV16 detection by qPCR method in relation to quantity and quality of DNA extracted from archival formalin fixed and paraffin embedded head and neck cancer tissues by three commercially available kits.
Author(s): Biesaga B, Janecka A, Mucha-Małecka A, Adamczyk A, Szostek S, Słonina D, Halaszka K, Przewoźnik M
Publication: J Virol Methods, 2016, Vol. 236, Page 157-63
PubMed ID: 27456982 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of DNA extraction method on the quantity and quality of DNA obtained from formalin-fixed paraffin-embedded (FFPE) head and neck cancer specimens and on the detection of human papillomavirus (HPV).
Conclusion of Paper
Although DNA yields were highest when DNA was extracted using the ExWAX kit, DNA purity was highest when extraction was with the QIAamp or ReliaPrep kit. Importantly, PCR success rates for β-actin were highest when DNA was extracted using the ReliaPrep kit and depended on A260/280 ratios when DNA was extracted using the QIAamp kit. HPV was detected by real-time PCR in eight of the 21 specimens, regardless of extraction method. Importantly, DNA yields were correlated between spectrophotometric and fluorometric methods but were significantly higher by spectrophotometry than fluoremetry. DNA yield and purity were not correlated with patient age, gender, or tumor stage or grade.
Studies
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Study Purpose
This study investigated the effects of DNA extraction method on the quantity and quality of DNA obtained from FFPE head and neck cancer specimens and on the detection of human papillomavirus (HPV). DNA was extracted from three to seven freshly cut 4 µm FFPE sections of squamous cell carcinoma of the mouth (5), tongue (5), tonsil (5), or oropharynx (6). Briefly, extraction with the Ex-WAX DNA Extraction kit involved washing with ethanol, overnight digestion at 50˚C, incubation with extraction solution, and precipitation. Extraction with the QIAamp DNA FFPE Tissue kit entailed deparaffinization with xylene and ethanol rehydration, overnight proteinase K digestion at 56˚C, demodification at 90˚C for 1 h, and column purification. DNA extraction with the ReliaPrepT FFPE gDNA Miniprep System involved incubation in 80˚C mineral oil for 1 min, addition of extraction solution and phase separation by centrifugation, overnight proteinase K digestion at 56˚C, demodification at 80˚C for 1 h, RNAse treatrment, phase separation, and column purification. DNA quality and yield were assessed by spectrophotometer, the QuantiFlour One dsDNA System, and by real-time PCR amplification of a 139 bp fragment of β-actin and a 81 bp fragment of HPV16 E6 gene.
Summary of Findings:
As determined by spectrophotometer, the ExWax kit resulted in significantly higher DNA yields than the QIAamp kit (16.9 µg versus 12.3 µg, P=0.0498) with intermediate yields obtained using the ReliaPrep kit (14.1 µg). The highest yields as determined by fluorometry also occurred with the ExWax kit (1.4 µg), but the yields by fluoremety were similar for the ReliaPrep and QIAamp kits (0.8 µg and 0.9 µg, respectively). As expected, the fluorometric yields were strongly to very strongly correlated with spectrophotometric yields (R=0.8471 for ExWAX, R=0.960 for QIAamp, and R=0.970 for ReliaPrep), but fluorometric yields were significantly lower, regardless of extraction method (P< 0.0001, all). DNA yield was not correlated with patient age, gender, or tumor stage or grade.
DNA purity as determined by spectrophotometric ratios was highest using the QIAamp kit and this kit also resulted in a higher percentage of specimens yielding A260/280 ratios between 1.8 and 2.0 than the ReliaPrep kit or ExWAX kit (85.7% versus 66.7% and 19.0%, respectively). The QIAamp kit also resulted in the highest OD260/230 ratios but a comparable percentage of specimens extracted with the QIAamp and ReliaPrep kits had OD 260/230 ratios in the acceptable range of 1.8-2.2 (57.1% and 61.9%, respectively). Only 9.5% of specimens extracted with the ExWAX kit had OD260/230 ratios in the acceptable range. The authors state that specimen purity was not correlated with patient age, gender, or tumor stage or grade.
β -actin amplification was successful in 20 of the 21 specimens extracted with the ReliaPrep kit and 17 of the 21 specimens when extracted with the QIAamp or ExWAX kits. β-actin failed to amplify in one of the specimens, regardless of extraction kit, and in two other specimens when extracted with ExWAX or QIAamp kits. Importantly, while DNA yield was not found to impact PCR success, the four specimens that failed to amplify after extraction with the QIAamp kit had significantly lower A260/280 ratios than the 17 for which amplification was observed (P< 0.0001). Interestingly, HPV was detected in the same eight specimens, regardless of extraction method, which was true even for those specimens in which β-actin failed to amplify.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Fluorometry DNA Real-time qPCR DNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Patient gender Female
Male
Preaquisition Patient age Not specified range
Real-time qPCR Specific Targeted nucleic acid β-actin
HPV
Analyte Extraction and Purification Analyte isolation method EX-WAX DNA Extraction Kit
QIAamp DNA FFPE Tissue kit
ReliaPrep FFPE gDNA Miniprep System
Preaquisition Prognostic factor Clinical stage T3N2
Clinical stage T4N1
Clinical stage T4N2
Clinical stage T4N3
Clinical stage T2N2