Quantitative reverse‐transcription PCR for miRNA model validation

Quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR) was used to profile the miRNAs identified as significant in OPSCC. The details of this experimental procedure have been described previously ¹⁷. Briefly, the RT reaction was performed with the High Capacity cDNA Reverse Transcription Kit (Life Technologies, Foster City, CA, USA). Each RT reaction included 100 ng of tumor RNA and a pool of RT primers for selected miRNAs and control RNAs. Quantitative PCR was performed with Power SYBR Green PCR Master Mix (Life Technologies) and specific PCR primers for selected miRNAs or control RNAs. miRNA raw profiling data for individual samples were normalized with four small RNA controls (SNORD48, SNORD47, RNA5‐8S5, and RNU6‐1). Specifically, the expression levels of the four small RNAs were averaged and used as the reference to control for sample variations during miRNA profiling analysis.

The expression of p16 protein was determined by immunohistochemistry as previously described ¹⁸. The expression profiles of E6 and E7 transcripts from six oncogenic HPV types were determined by qRT‐PCR, including types 16, 18, 33, 39, 56, and 59. The details of the HPV assays and the experimental protocol have been described previously ¹⁸. In brief, primer sequences for the assays were selected from the E6 and E7 coding regions of the high‐risk HPV genomes. The expression profiles of GAPDH and β‐actin were used as reference controls for data normalization.