From each FFPE sample, five to seven slices (10 µm) were used for DNA isolation, utilizing a commercial kit (NucleoSpin® DNA FFPE XS, Macherey–Nagel, Dueren, Germany) according to the manufacturer’s instructions. The concentration of the isolated DNA was measured using NanoPhotometer® N60 (Implen GmbH, Munich, Germany), and the quality was validated by PCR using primers generating 99 bp long beta-actin fragments [40 (link),41 (link)].
For HPV DNA detection, PCR was performed using short primers suitable for FFPE tissue samples GP5/6 and SPF 10, generating approximately 142 bp and 65 bp long PCR products, respectively [42 (link),43 (link)]. PCR amplification was performed as previously described [40 (link),44 ] and 10 μL of amplified PCR products were run on 3% agarose gels (Sigma Aldrich, St. Louis, MO, USA). A sample was considered to be HPV+ if either the GP5/6 or SPF10 PCR was positive. Additionally, HPV16+ samples were distinguished using a supplementary primer pair generating a shorter DNA sequence of 98 bp, as previously described [45 (link)].
For HPV DNA detection, PCR was performed using short primers suitable for FFPE tissue samples GP5/6 and SPF 10, generating approximately 142 bp and 65 bp long PCR products, respectively [42 (link),43 (link)]. PCR amplification was performed as previously described [40 (link),44 ] and 10 μL of amplified PCR products were run on 3% agarose gels (Sigma Aldrich, St. Louis, MO, USA). A sample was considered to be HPV+ if either the GP5/6 or SPF10 PCR was positive. Additionally, HPV16+ samples were distinguished using a supplementary primer pair generating a shorter DNA sequence of 98 bp, as previously described [45 (link)].
Free full text: Click here
