Characterization of Human Salivary Extracellular RNA by Next-generation Sequencing.

BACKGROUND: It was recently discovered that abundant and stable extracellular RNA (exRNA) species exist in bodily fluids. Saliva is an emerging biofluid for biomarker development for noninvasive detection and screening of local and systemic diseases. Use of RNA-Sequencing (RNA-Seq) to profile exRNA is rapidly growing; however, no single preparation and analysis protocol can be used for all biofluids. Specifically, RNA-Seq of saliva is particularly challenging owing to high abundance of bacterial contents and low abundance of salivary exRNA. Given the laborious procedures needed for RNA-Seq library construction, sequencing, data storage, and data analysis, saliva-specific and optimized protocols are essential. METHODS: We compared different RNA isolation methods and library construction kits for long and small RNA sequencing. The role of ribosomal RNA (rRNA) depletion also was evaluated. RESULTS: The miRNeasy Micro Kit (Qiagen) showed the highest total RNA yield (70.8 ng/mL cell-free saliva) and best small RNA recovery, and the NEBNext library preparation kits resulted in the highest number of detected human genes [5649-6813 at 1 reads per kilobase RNA per million mapped (RPKM)] and small RNAs [482-696 microRNAs (miRNAs) and 190-214 other small RNAs]. The proportion of human RNA-Seq reads was much higher in rRNA-depleted saliva samples (41%) than in samples without rRNA depletion (14%). In addition, the transfer RNA (tRNA)-derived RNA fragments (tRFs), a novel class of small RNAs, were highly abundant in human saliva, specifically tRF-4 (4%) and tRF-5 (15.25%). CONCLUSIONS: Our results may help in selection of the best adapted methods of RNA isolation and small and long RNA library constructions for salivary exRNA studies.

Cancer Salivary Biomarkers for Tumours Distant to the Oral Cavity.

The analysis of saliva as a diagnostic approach for systemic diseases was proposed just two decades ago, but recently great interest in the field has emerged because of its revolutionary potential as a liquid biopsy and its usefulness as a non-invasive sampling method. Multiple molecules isolated in saliva have been proposed as cancer biomarkers for diagnosis, prognosis, drug monitoring and pharmacogenetic studies. In this review, we focus on the current status of the salivary diagnostic biomarkers for different cancers distant to the oral cavity, noting their potential use in the clinic and their applicability in personalising cancer therapies.

Non-coding RNAs in saliva: emerging biomarkers for molecular diagnostics.

Saliva is a complex body fluid that comprises secretions from the major and minor salivary glands, which are extensively supplied by blood. Therefore, molecules such as proteins, DNA, RNA, etc., present in plasma could be also present in saliva. Many studies have reported that saliva body fluid can be useful for discriminating several oral diseases, but also systemic diseases including cancer. Most of these studies revealed messenger RNA (mRNA) and proteomic biomarker signatures rather than specific non-coding RNA (ncRNA) profiles. NcRNAs are emerging as new regulators of diverse biological functions, playing an important role in oncogenesis and tumor progression. Indeed, the small size of these molecules makes them very stable in different body fluids and not as susceptible as mRNAs to degradation by ribonucleases (RNases). Therefore, the development of a non-invasive salivary test, based on ncRNAs profiles, could have a significant applicability to clinical practice, not only by reducing the cost of the health system, but also by benefitting the patient. Here, we summarize the current status and clinical implications of the ncRNAs present in human saliva as a source of biological information.

RNA Sequencing Analysis of Salivary Extracellular RNA.

Salivary biomarkers for disease detection, diagnostic and prognostic assessments have become increasingly well established in recent years. In this chapter we explain the current leading technology that has been used to characterize salivary non-coding RNAs (ncRNAs) from the extracellular RNA (exRNA) fraction: HiSeq from Illumina® platform for RNA sequencing. Therefore, the chapter is divided into two main sections regarding the type of the library constructed (small and long ncRNA libraries), from saliva collection, RNA extraction and quantification to cDNA library generation and corresponding QCs. Using these invaluable technical tools, one can identify thousands of ncRNA species in saliva. These methods indicate that salivary exRNA provides an efficient medium for biomarker discovery of oral and systemic diseases.