Background: Small RNAs such as microRNA (miRNAs) are generally released into circulation during times of disease, stress and homeostatic imbalances. The function of these extracellular miRNAs remains to be determined however their expression patterns in the blood is recognised as a blue print for a particular disease type. Oral cancers are on the rise in developed countries with early diagnosis being the key to overall survival. Our lab has identified five serum miRNAs as potential biomarkers in oral cancers but one of the major issues is normalising miRNA fold expression after quantitative PCR (qPCR). To bypass this standard fold normalisation we developed a qPCR assay to measure the exact copy numbers of serum miRNAs.

Aim: In this study, we present an absolute quantitative PCR approach to accurately measure serum miRNAs. Using this method we then assessed the potential of serum miRNAs as biomarkers for oral cancers.

Methods: Synthetic RNA oligonucleotides representing specific miRNAs were used as templates to generate a standard concentration curve (10 to X copies). We then determined the exact copy numbers for several miRNAs in stage 1 and 2 oral cancer (n=20) versus normal (n=20) serum samples.

Results: Absolute qPCR quantitation was able to determine the exact copy numbers of several specific miRNAs in serum. These miRNAs were significantly higher in the oral cancer sera when compared to healthy patients. Using this absolute qPCR strategy, we can derive an exact copy number threshold that may be indicative for a positive diagnosis for oral cancers.

Discussion: Using absolute qPCR, we were able to determine the exact number of miRNAs in oral cancer serum and to assess their potential as early biomarkers. This approach does not rely on the identification of a reference miRNA for fold normalisation thus expanding its utility to measure any miRNAs in serum.