Abstract

Challenges in the Deployment of Saliva in Diagnostics Development

David Wong, Director, University of California Los Angeles

Extracellular RNAs (exRNAs) in biofluids have sparked considerable interests in their use as disease-specific diagnostic and prognostic biomarkers. In the last decade, it has been demonstrated the potential use of salivary RNA for detecting various local and systemic diseases such as oral cancer, Sjögren syndrome, pancreatic cancer and breast cancer. The ability of sequence-characterized salivary exRNA by using next-generation sequencing can further strengthen the advantages of using saliva as a clinical diagnostic biofluid for biomarker discovery.  Compared with other bodily fluids, saliva can be collected easily and noninvasively. However, low RNA abundance, small sample volumes, high fragmentation of RNA and lots of bacterial contents create challenges for downstream RNA sequencing assays. We systematically tested RNA isolation efficiency with six commercially available kits with optimized protocols and compared their performance on saliva samples mimicking clinical features. Highly sensitive RiboGreen assays were used to quantify total RNA yield and qPCR/ddPCR assays were applied to determine the efficiency of long and small RNA isolation from each kit. The highest RNA yield was 20-80 ng RNA/mL cell free saliva resulted from miRNeasy kit. To evaluate different RNA-Seq library construction methods, we used multiple commercially available kits for long and small RNA library preparation. The NEB library preparation kits resulted in the highest number of human genes and small RNAs species. Generally, 23-36% of reads from long RNA library are from bacterial ribosomal RNA. To increase the sensitivity in detecting human transcripts, a selective removal of bacterial ribosomal RNA (rRNA) protocol was optimized. The analysis results showed much fewer microbial reads (0.8%-4.6%) and at least 48% more genes can be detected in rRNA-depleted samples compared to the control. This observation indicated that the rRNA removal step could improve the comprehensiveness of human