DNA extraction and HPV detection by PCR

Total DNA was extracted from biopsies using the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA) following the manufacturer's instructions: 15–25 mg tissue sections were applied to a clean single-edge blade using strict aseptic technique to prevent contamination or cross-contamination of samples. The samples were digested overnight at 55°C in the lysis Buffer AL containing protease K (Qiagen, Valencia, CA), followed by precipitation of DNA with absolute ethanol in a spin column. The DNA was eluted with Buffer AE (Qiagen, Valencia, CA). DNA purity and concentration were quantified using a BioPhotometer (Eppendorf, Hauppauge, NY). Two sets of primers specific to the E6 oncogene were used to detect HPV types 16 and 18 DNA (p16F/p16R and p18F/p18R, respectively, Table 1) [19–21]. The expected size for both PCR products was 140 bp. DNA integrity was determined by amplification of the human β-globin gene prior to HPV PCR assay, as described in the literature [19,22] using Globin-1 and -2 primers (Table 1). The expected size for the PCR product was 268 bp.

The PCR assays were performed as described by others [21,22] with minor modifications. All reagents were from Life Technologies, Carlsbad, CA, unless otherwise specified. Briefly, test sample DNA (10 to 200 ng) was added to a mixture containing dNTP (0.2 mM each), Taq polymerase (0.04 U), reaction buffer (1X), MgCl₂ (1.5 mM), reverse and forward primers for HPV 16 or HPV 18 (0.5 μM each; Eurofins MWG, Les Ulis, France); PCR-quality water was used to reach a final volume of 25 μL. To avoid false positive and/or negative results, a negative control (no template DNA) and an HPV-positive DNA sample were included in each assay. PCR amplification was performed using a DNA Thermocycler (MJ Research, Waltham, MA, USA), as follows: 5 min at 94°C, 40 cycles (94°C, 1 min; 65°C, 1 min; 72°C, 2 min) and a final 7 min extension period at 72°C.

As a control of the PCR product size, 12 μl of PCR product were loaded on a 2% 0.5X TBE buffer agarose gel, stained with 0.3 mg/mL ethidium bromide, and photographed by UV transillumination with a KODAK EDAS 290. The molecular weight of the PCR products was determined by comparison to DNA molecular weight marker XIV (Roche Diagnostics, Mannheim, Germany).