Journal
CLINICAL CHEMISTRY
Volume 64, Issue 7, Pages 1085-1095Publisher
AMER ASSOC CLINICAL CHEMISTRY
DOI: 10.1373/clinchem.2017.285072
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Funding
- Public Health Service (PHS) grants from the National Institute of Health (NIH) [UH3 TR000923, R90 DE022734]
- 2017 Debbie's Dream Foundation - American Association for Cancer Research (AACR) Gastric Cancer Research Fellowship [17-40-41-KACZ]
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BACKGROUND: It was recently discovered that abundant and stable extracellular RNA (exRNA) species exist in bodily fluids. Saliva is an emerging biofluid for bio-marker 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 (RPKIvI)] and small RNAs [482696 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. (C) 2018 American Association for Clinical Chemistry
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