A method to detect microRNAs and viral RNAs in human saliva
- School of Biomedical Engineering, Faculty of Engineering and IT, University of Technology Sydney.
- Sydney Head and Neck Cancer Institute, Sydney Cancer Centre, Royal Prince Alfred Hospital.
The field of salivary diagnostics is growing because of its non-invasive collection and its potential as a biological fluid for biomarker discovery studies. Our lab is interested in using non-coding RNAs found in saliva as biomarkers for the early detection of oral cancer. This study will provide a robust methodology for the isolation of total RNA from the saliva of patients with OC. We then demonstrated that the RNA can be used to detect a range of long and short ncRNAs using RT-qPCR. Our approach utilises 1mL of whole saliva collected from 12 control volunteers and 10 patients with oral cancer. Total RNA was extracted from supernatant saliva utilising a liquid-based guanidine isothiocyanate reagent. This modified approach yielded high quantities (200ng/μl) of RNA material. We found that cell-free salivary supernatant proved a more consistent medium for total RNA yields than whole saliva or cell pellets. With this total RNA, we were able to detect specific species of microRNAs such as miR-21 and other mRNAs. Furthermore, we were able to detect viral RNA in the saliva. With this approach, we hope to develop an assay for the detection of aberrant miRNAs or HPV using the saliva of oral cancer patients. In summary, we have optimised a methodology which can provide sufficient quantities of total RNA from human spit. This material can be utilised for diagnostic purposes such as the detection of miRNA or viral biomarkers. It is a cost-effective method which most laboratories can adopt and only uses minimal amounts (1mL) of saliva.