Division of Newborn Medicine, Department of Pediatrics, The Floating Hospital for Children at Tufts Medical Center, Medford, MA, USA.
Neonatology. 2012;101(1):55-60. doi: 10.1159/000328026. Epub 2011 Jul 26.
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.
The aim of this study was to develop techniques to enhance extraction of cell-free RNA from neonatal salivary supernatant.
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.
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.
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.
唾液上清液的基因表达谱分析正在成为实时、系统、生物学信息的新的重要来源。然而，现有的技术阻止了从小量新生儿唾液上清液中提取 RNA。
本研究旨在开发从新生儿唾液上清液中增强提取无细胞 RNA 的技术。
从（36-41 周妊娠）新生儿（n = 13）中连续收集两个唾液样本（10-100 μl）并进行稳定化处理。使用两种改良的提取技术：Qiagen RNAprotect® Saliva Mini Kit（方法 1）和 QIAamp Viral RNA Mini Kit（方法 2）从唾液上清液中提取总 RNA。对提取的唾液样本进行 GAPDH 的定量 RT-PCR 扩增。对每个方案的 RNA 产量进行平均阈值循环（Ct）水平的统计分析。对新生儿全唾液和上清液（n = 3）进行配对微阵列分析，以区分这些生物层之间的基因表达差异。
成功地从所有唾液上清液样本中提取并扩增了 mRNA。与方法 1 相比，方法 2 的提取量更多（p = 0.008）。全唾液和上清液的配对基因表达分析存在 7.5%的差异。在上清液中统计学上显著上调的基因突出了 16 个在全唾液中未见的独特生物学功能。相反，全唾液中只有两个生物学功能是独特的。
新生儿无细胞唾液上清液 mRNA 可轻松提取并用于下游应用。这些技术增强使新生儿唾液转录组的诊断潜力得到进一步探索成为可能。
