Optimal Techniques for mRNA Extraction from Neonatal Salivary Supernatant

Abstract

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.

The Neonatal Salivary Transcriptome

“…More recently, Pandit and colleagues (2013) have modified an existing Qiagen protocol (QIAzol), to provide investigators with a robust, cost-effective alternative to other commercially available RNA extraction kits. By comparison, published reports in the newborn have successfully used Qiagen's RNAProtect Saliva Solution on multiple downstream transcriptomic platforms (Maron et al , 2012bDietz et al 2012). Thus, investigators have options when considering salivary collection and extraction kits and should consider study design, location and costs when choosing a product or protocol.…”

“…Saliva is collected in the syringe for 20 -30 sec and immediately stabilized at the bedside. Samples may then be left at room temperature, 4˚C or frozen at -20˚C or -80˚C pending processing and manufacturer's guidelines (Dietz et al 2012). Most commercially available collection kits allow for samples to sit at room temperature for days to weeks without any risk of RNA degradation.…”

“…Sponges are also available for saliva collection in the newborn, but investigators must take the additional step of centrifugation to retrieve the saliva sample. It should be noted that in the neonatal population, modifications to existing protocols might need to be made to account for the limited salivary volumes available (Table 1) (Maron 2011;Dietz et al 2012). Most salivary collection kits recommend 1 mL of saliva for downstream RNA extraction and analysis.…”

Other Body Fluids as Non-invasive Sources of Cell-Free DNA/RNA

“…Neonatal studies conducted by Deitz et al [35] compared gene expression differences between whole saliva with cell-free salivary supernatant through microarray and RT-qPCR analysis. Total cfRNA was successfully extracted from all neonatal salivary supernatant samples, even from volumes as low as 10 μl (range 10-100 μl).…”

Comprehensive Analysis of Genes Expressed by Rare Microchimeric Fetal Cells in the Maternal Mouse Lung1

“…All samples were run on a 7900 model sequence detector with TaqMan One-Step RT-PCR Master Mix reagents kit (Applied Biosystems, Foster City, CA) as previously described [22].…”