Abstract
Non-invasive techniques to monitor and diagnose neonates, particularly those born prematurely, are a long-sought out goal of Newborn Medicine. In recent years, technical advances, combined with increased assay sensitivity, have permitted the high-throughput analysis of multiple biomarkers simultaneously from a single sample source. Multiplexed transcriptomic and proteomic platforms, along with more comprehensive assays such as RNASeq, allow for interrogation of ongoing physiology and pathology in unprecedented ways. In the fragile neonatal population, saliva is an ideal biofluid to assess clinical status serially and offers many advantages over more invasively obtained blood samples. Importantly, saliva samples are amenable to analysis on emerging proteomic and transcriptomic platforms, even at quantitatively limited volumes. However, biomarker targets are often degraded in human saliva, and as a mixed source biofluid containing both human and microbial targets, saliva presents unique challenges for the investigator. Here, we provide insight into technical considerations and protocol optimizations developed in our laboratory to quantify and discover neonatal salivary biomarkers with improved reproducibility and reliability. We will detail insights learned from years of experimentation on neonatal saliva within our laboratory ranging from salivary collection techniques to processing to downstream analyses, highlighting the need for consistency in approach and a global understanding of both the potential benefits and limitations of neonatal salivary biomarker analyses. Importantly, we will highlight the need for robust and stringent research in this population to provide the field with standardized approaches and workflows to impact neonatal care successfully.
Highlights
Neonatologists have recognized that saliva provides a window into their vulnerable patients’ physiology and pathophysiology [1, 2]
While initial studies focused on quantitative cortisol levels with corresponding stress response [3, 4], more recent studies have demonstrated that salivary analyses can provide insight into development through both global and targeted gene and/or protein expression analyses [5,6,7]
As the field continues to evolve toward clinical translation, it is imperative that researchers and clinicians recognize the potential benefits and limitations of saliva as a non-invasive biofluid for biomarker discovery and neonatal assessment
Summary
Neonatologists have recognized that saliva provides a window into their vulnerable patients’ physiology and pathophysiology [1, 2]. Comparative analyses of salivary profiles between infants affected by a variety of disorders with unaffected control infants allows us to identify novel biomarkers with the use of large-scale screening platforms described in this article, such as the NOuRISH platform (A) or assess the accuracy of known biomarkers, such as the SPITSS Trial (B) in making a diagnosis Both proteins and gene targets (mRNA) are amenable to these approaches. The investigator is limited to gene expression analyses in lieu of a more comprehensive analysis of regulatory RNAs. The second technical issue confronted with RNASeq analysis of neonatal saliva is the potential for increased alternative splicing of transcripts, limiting read alignment to the human genome.
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