Performing discovery-driven neonatal research by transcriptomic analysis of routinely discarded biofluids.

OBJECTIVE: To perform discovery-driven research on the neonatal salivary and cord blood transcriptomes. METHODS: Two separate cohorts of infants were enrolled in this study. In one, cord blood (n = 10) and in the other, saliva samples (n = 10) were collected at term gestation. Total RNA was extracted, amplified and hybridized onto Affymetrix HG U133a gene expression microarrays. Following normalization, genes expressed in the highest quintile (≥ 80%) across all subjects in each biofluid were analyzed with Ingenuity Pathway Analysis. Over-represented pathways relating to organ specific development and physiological functions in the newborn were explored. RESULTS: There were 303 genes in neonatal saliva and 282 genes in umbilical cord blood that met statistical criteria. Of these, 114 were common to both biofluids. Pathway analyses revealed the important roles of redox balance, cellular proliferation, and smooth muscle relaxation. In blood, hematopoiesis and immune response pathways predominated. In saliva, pathways associated with the gastrointestinal system were highlighted. CONCLUSIONS: Neonatal cord blood and saliva provide a wealth of transcriptomic information. These normally discarded biofluids should be considered an important source of real-time gene expression data that may elucidate key pathways in neonatal physiology and pathology.

Neuropeptide Y2 receptor (NPY2R) expression in saliva predicts feeding immaturity in the premature neonate.

BACKGROUND: The current practice in newborn medicine is to subjectively assess when a premature infant is ready to feed by mouth. When the assessment is inaccurate, the resulting feeding morbidities may be significant, resulting in long-term health consequences and millions of health care dollars annually. We hypothesized that the developmental maturation of hypothalamic regulation of feeding behavior is a predictor of successful oral feeding in the premature infant. To test this hypothesis, we analyzed the gene expression of neuropeptide Y2 receptor (NPY2R), a known hypothalamic regulator of feeding behavior, in neonatal saliva to determine its role as a biomarker in predicting oral feeding success in the neonate. METHODOLOGY/PRINCIPAL FINDINGS: Salivary samples (n = 116), were prospectively collected from 63 preterm and 13 term neonates (post-conceptual age (PCA) 26 4/7 to 41 4/7 weeks) from five predefined feeding stages. Expression of NPY2R in neonatal saliva was determined by multiplex RT-qPCR amplification. Expression results were retrospectively correlated with feeding status at time of sample collection. Statistical analysis revealed that expression of NPY2R had a 95% positive predictive value for feeding immaturity. NPY2R expression statistically significantly decreased with advancing PCA (Wilcoxon test p value<0.01), and was associated with feeding status (chi square p value  =  0.013). CONCLUSIONS/SIGNIFICANCE: Developmental maturation of hypothalamic regulation of feeding behavior is an essential component of oral feeding success in the newborn. NPY2R expression in neonatal saliva is predictive of an immature feeding pattern. It is a clinically relevant biomarker that may be monitored in saliva to improve clinical care and reduce significant feeding-associated morbidities that affect the premature neonate.

Optimal techniques for mRNA extraction from neonatal salivary supernatant.

BACKGROUND: Gene expression profiling of the salivary supernatant is emerging as a new and important source of real-time, systemic, biological information. However, existing technologies prevent RNA extraction of small quantities found in neonatal salivary supernatant. OBJECTIVE: The aim of this study was to develop techniques to enhance extraction of cell-free RNA from neonatal salivary supernatant. METHODS: Two saliva samples (10-100 µl) were serially collected from newborns (36-41 weeks' gestation) (n = 13) and stabilized. Total RNA was extracted from salivary supernatant with the use of two modified extraction techniques: Qiagen RNAprotect® Saliva Mini Kit (method 1) and the QIAamp Viral RNA Mini Kit (method 2). Quantitative RT-PCR amplification for GAPDH was performed on extracted salivary samples. Statistical analyses were performed on mean threshold cycle (Ct) levels to compare RNA yield from each protocol. Paired microarray analyses were made between neonatal whole saliva and supernatant (n = 3) to discern gene expression differences between these biolayers. RESULTS: mRNA was successfully extracted and amplified from all salivary supernatant samples. Extraction with method 2 yielded more RNA than with method 1 (p = 0.008). There was a 7.5% discordance between paired gene expression analyses for whole saliva and supernatant. Genes that were statistically significantly upregulated in supernatant highlighted 16 distinct biological functions not seen in whole saliva. Conversely, only two biological functions were unique to whole saliva. CONCLUSION: Neonatal cell-free salivary supernatant mRNA may be readily extracted and utilized on downstream applications. These technical enhancements allow for further exploration of the diagnostic potential of the neonatal salivary transcriptome.