1. Characterization of Human Salivary Extracellular RNA by Next-generation Sequencing
- Author
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Blanca Majem, Feng Li, Karolina Elżbieta Kaczor-Urbanowicz, David Chia, Tristan Grogan, Kyoung-Mee Kim, So Young Kang, David Elashoff, David T.W. Wong, Su Mi Kim, Shannon Liu Rao, Sung Kim, Jie Sun, Hsien-Chun Lo, Xinshu Xiao, Yong Kim, and Kikuye Koyano
- Subjects
0301 basic medicine ,Small RNA ,DNA, Complementary ,Sequence analysis ,Medical Biotechnology ,Clinical Sciences ,Clinical Biochemistry ,Medical Biochemistry and Metabolomics ,Biology ,Article ,03 medical and health sciences ,Complementary ,microRNA ,Genetics ,Humans ,Saliva ,General Clinical Medicine ,Sequence Analysis, RNA ,Biochemistry (medical) ,RNA ,DNA ,Ribosomal RNA ,030104 developmental biology ,Biochemistry ,Transfer RNA ,RNA extraction ,Extracellular Space ,Sequence Analysis ,Biotechnology ,Extracellular RNA - Abstract
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.
- Published
- 2018