title: Optimal techniques for mRNA extraction from neonatal salivary supernatant.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Division of Newborn Medicine, Department of Pediatrics, The Floating Hospital for Children at Tufts Medical Center, Medford, MA, USA.
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Dietz
authors.forename: Kirby L
authors.initials: KL
authors.lastname: Johnson
authors.forename: Heather C
authors.initials: HC
authors.lastname: Wick
authors.forename: Diana W
authors.initials: DW
authors.lastname: Bianchi
authors.forename: Jill L
authors.initials: JL
authors.lastname: Maron
mesh_terms: D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D005865:Gestational Age; D006801:Humans; D007231:Infant, Newborn; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012463:Saliva / Q000737:chemistry*; D014158:Transcription, Genetic
chemical_list: D012333:RNA, Messenger