Total RNA was purified from three pooled sections of the same tissue block using the Pure Link FFPE Total RNA Isolation Kit (Invitrogen, Carlsbad, CA) as described previously [41 (link)]. RT-PCR was carried out using the QuantiTect Virus Kit (Qiagen, Hilden, Germany) in a total volume of 25 μL containing 5 μL of 5xQuantiTect Virus Mastermix, 0.25 μL of 100xQuantiTect Virus RT Mix, 0.4 μM of each oligonucleotide, and 1 μL RNA as described previously [42 (link)]. The HPV type-specific E6*I mRNA assay developed for 20 HR- or pHR-HPV types was applied for the detection of viral transcripts. The assay amplifies a 65–75 base pair amplicon of HPV and an 81 base pair amplicon of ubiquitin C (ubC) cDNA.
The HPV RNA analysis was performed at the German Cancer Research Center laboratories in Heidelberg, Germany.
The HPV RNA analysis was performed at the German Cancer Research Center laboratories in Heidelberg, Germany.
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