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.

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.

Fig. 1

Schematic of neonatal salivary acquisition. Step 1: 1-ml syringe is first modified by removing the endcaps; step 2: the plunger is removed, saved and set aside; step 3: the modified syringe is attached to low wall suction and salivary sample is gently collected from the neonate's oropharynx; step 4: the sample is immediately placed into 1 ml of RNAProtect Saliva Solution; step 5: the sample is vortexed for 20 s; step 6: the sample is placed immediately on ice; step 7: the sample is brought back to the laboratory for further processing.