Analysis of HPV Transcription by RPA

Human papillomavirus (HPV) transcription is a complex process using multiple promoters, splices, and polyadenylation sites to create polycistronic transcripts capable of encoding the known and putative viral genes at the correct time and place throughout the differentiation-dependent life cycle. The ribonuclease protection assay (RPA) provides a flexible and convenient tool to study the behavior of HPV transcripts under a variety of cellular conditions and treatments, or in response to genetic mutations. Using a known cloned DNA as a template, an antisense RNA probe is generated and hybridized to the sample RNA. After digestion with ribonucleases (RNases), the fragments of the probe protected by the sample are examined by gel electrophoresis. With the proper design of the probe template, information about promoter usage, splicing, transcript levels, and other parameters can be accurately, simply, and quantitatively measured throughout the HPV life cycle.