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

• Published in: Journal of virological methods 2016-10, Vol.236, p.157-163
• Main Authors: Biesaga, Beata, Janecka, Anna, Mucha-Małecka, Anna, Adamczyk, Agnieszka, Szostek, Sława, Słonina, Dorota, Halaszka, Krzysztof, Przewoźnik, Marcin
• Format: Article
• Language: English
• Subjects: DNA quality; DNA, Viral - genetics; DNA, Viral - isolation & purification; FFPE tissue; Head and Neck Neoplasms - diagnosis; Head and Neck Neoplasms - virology; HPV detection; Human papillomavirus 16; Human papillomavirus 16 - genetics; Human papillomavirus 16 - isolation & purification; Humans; Molecular Diagnostic Techniques - methods; Papillomavirus Infections - complications; Papillomavirus Infections - diagnosis; Paraffin Embedding; Pathology, Molecular - methods; Reagent Kits, Diagnostic; Real-Time Polymerase Chain Reaction - methods; Tissue Fixation
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2016.07.021

•HPV16 detection by qPCR is partly dependent on quality of DNA extracted from archival tissues.•All HPV16 positive samples extracted with Promega kit were β-actin amplifiable.•For DNA isolation from archival tissues we recommend Promega kit. The aim of the present study was to compare HPV16 detection by quantitative polymerase chain reaction (qPCR) in relation to the quantity and quality of DNA isolated from 21 formalin fixed and paraffin embedded (FFPE) head and neck cancer tissues by three commercially available kits: EX-WAX™ DNA Extraction Kit (M) (Merck Millipore, Darmstadt, Germany), QIAamp® DNA FFPE Tissue (Q) (Qiagen, Hilden, Germany) and ReliaPrep™ FFPE gDNA Miniprep System (P) (Promega, Madison, USA). Quantity of extracted DNA was assessed spectrophometrically and fluorometrically. Its quality was analyzed using A260/280 and A260/230 ratios and the β-actin fragment amplifiability in qPCR. HPV16 presence was detected by qPCR, using specific primers and TaqMan probe. HPV infection was found in 8 DNA samples extracted with M kit (38.1%) and in 7 (33.3%) isolated with Q and P kits. Three samples from M and Q kits were characterized by HPV16 positivity and lack of β-actin amplifiability. They had significantly lower A260/280 ratio (M: 1.6±0.0, p=0.044 and Q: 1.7±0.0, p=0.016) compared to samples with both fragments amplification (M: 1.7±0.0 and Q: 1.9±0.0). Therefore, for HPV detection by qPCR in FFPE tissues we recommend ReliaPrep™ FFPE gDNA Miniprep System.