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.
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.
Authors: Christopher Wright; Donald Bergstrom; Hongyue Dai; Matthew Marton; Mark Morris; George Tokiwa; Yanqun Wang; Thomas Fare Journal: Clin Chem Date: 2007-12-18 Impact factor: 8.327
Authors: Jill L Maron; Kirby L Johnson; David M Rocke; Michael G Cohen; Albert J Liley; Diana W Bianchi Journal: Clin Chem Date: 2009-12-03 Impact factor: 8.327
Authors: Lei Zhang; James J Farrell; Hui Zhou; David Elashoff; David Akin; No-Hee Park; David Chia; David T Wong Journal: Gastroenterology Date: 2009-11-18 Impact factor: 22.682
Authors: Enders K O Ng; Nancy B Y Tsui; Nicole Y L Lam; Rossa W K Chiu; Simon C H Yu; S C Cesar Wong; Elena S F Lo; Timothy H Rainer; Philip J Johnson; Y M Dennis Lo Journal: Clin Chem Date: 2002-08 Impact factor: 8.327
Authors: Ze Tian; Nathan Palmer; Patrick Schmid; Hui Yao; Michal Galdzicki; Bonnie Berger; Erxi Wu; Isaac S Kohane Journal: PLoS One Date: 2009-04-17 Impact factor: 3.240
Authors: Peterson T Haak; Julia V Busik; Eric J Kort; Maria Tikhonenko; Nigel Paneth; James H Resau Journal: Neonatology Date: 2008-09-18 Impact factor: 4.035
Authors: Jill L Maron; Jooyeon S Hwang; Subash Pathak; Robin Ruthazer; Ruby L Russell; Gil Alterovitz Journal: J Pediatr Date: 2015-02 Impact factor: 4.406
Authors: Stephanie Pritchard; Heather C Wick; Donna K Slonim; Kirby L Johnson; Diana W Bianchi Journal: Biol Reprod Date: 2012-08-23 Impact factor: 4.285
Authors: Joy Gardner; Penny A Rudd; Natalie A Prow; Essia Belarbi; Pierre Roques; Thibaut Larcher; Lionel Gresh; Angel Balmaseda; Eva Harris; Wayne A Schroder; Andreas Suhrbier Journal: PLoS One Date: 2015-10-08 Impact factor: 3.240