Technical Considerations and Protocol Optimization for Neonatal Salivary Biomarker Discovery and Analysis

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.

Metabolomics to Diagnose Oxidative Stress in Perinatal Asphyxia: Towards a Non-Invasive Approach

“…However, technological advances enable the analysis of small sample volumes ( Table 2 ). Yen et al [ 23 ] state that suctioning of the mouth typically yields 10–50 μL neonatal saliva, whereas sponges and wick applicators may yield slightly higher volumes, directly correlated to collection time. Oxidative stress markers glutathione [ 24 ], malondialdehyde [ 24 , 25 , 26 , 27 ] catalase, protein carbonyls, glutathione peroxidase, and 8-hydroxy-2′-deoxyguanosine [ 27 ], as well as isoprotanes, isofuranes, neuroprostanes, and neurofuranes [ 28 ] have all been measured in saliva from adults.…”

Sex-specific inflammatory and white matter effects of prenatal opioid exposure: a pilot study

“…Saliva was collected at least 1 hour from feeding to minimize milk contamination using previously described techniques. 24 , 25 Briefly, neonates’ mouths were gently suctioned using a 1 mL insulin syringe (Becton, Dickinson and Company, Franklin Lakes, NJ) attached to the low-pressure wall suction for approximately 15 to 30 seconds. Whole saliva was immediately placed in Eppendorf tubes pre-filled with 250 μL RNAprotect Saliva Reagent (Qiagen, Hilden, Germany) to minimize RNA degradation, then vortexed and stored at 4°C for at least 48 hours but no more than 28 days pending RNA extraction.…”

Proposal for a 6‐step approach for differential diagnosis of neonatal erythroderma

“…Although in most cases in this review genetic analysis was performed on blood samples, collecting material via non‐invasive methods are important to consider as a future alternative in patients with NE. Collecting saliva in neonates with buccal swabs for DNA and RNA sampling seem very promising, but different techniques herein determine optimal outcome 124 …”

“…Collecting saliva in neonates with buccal swabs for DNA and RNA sampling seem very promising, but different techniques herein determine optimal outcome. 124…”