Center of Periodontology Studies, Jalan Hospital, Universiti Teknologi MARA, Sungai Buloh, Malaysia.
Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New Zealand.
J Periodontal Res. 2018 Jun;53(3):369-377. doi: 10.1111/jre.12522. Epub 2017 Dec 27.
The salivary transcriptome may present as a readily available and non-invasive source of potential biomarkers. The development of chronic periodontitis is determined by individual patient susceptibility; hence, the aim of this study was to determine the potential of the salivary transcriptome as a biomarker of disease susceptibility using chronic periodontitis as an example.
Using an Oragene RNA kit, the total RNA was purified from the saliva of 10 patients with chronic periodontitis and 10 patients without chronic periodontitis. The quantity and quality of the total RNA was determined, and a measure of gene expression via cDNA was undertaken using the Affymetrix microarray system. The microarray profiling result was further validated by real-time quantitative polymerase chain reaction.
Spectrophotometric analysis showed the total RNA purified from each participant ranged from 0.92 μg/500 μL to 62.85 μg/500 μL. There was great variability in the quantity of total RNA obtained from the 2 groups in the study with a mean of 10.21 ± 12.71 μg/500 μL for the periodontitis group and 15.97 ± 23.47 μg/500 μL for the control group. Further the RNA purity (based on the A /A ratio) for the majority of participants (9 periodontitis and 6 controls) were within the acceptable limits for downstream analysis (2.0 ± 0.1). The study samples, showed 2 distinct bands at 23S (3800 bp) and 16S (1500 bp) characteristic of bacterial rRNA. Preliminary microarray analysis was performed for 4 samples (P2, P6, H5 and H9). The percentage of genes present in each of the 4 samples was not consistent with about 1.8%-18.7% of genes being detected. Quantitative real-time polymerase chain reaction confirmed that the total RNA purified from each sample was mainly bacterial RNA (Uni 16S) with minimal human mRNA.
This study showed that minimal amounts of human RNA were able to be isolated from the saliva of patients with periodontitis as well as controls. Further work is required to enhance the extraction process of human mRNA from saliva if the salivary transcriptome is to be used in determining individual patient susceptibility.
唾液转录组可能是一种易于获得且非侵入性的潜在生物标志物来源。慢性牙周炎的发生发展取决于个体患者的易感性；因此，本研究旨在以慢性牙周炎为例，探讨唾液转录组作为疾病易感性生物标志物的潜力。
采用 Oragene RNA 试剂盒，从 10 例慢性牙周炎患者和 10 例无慢性牙周炎患者的唾液中提取总 RNA。测定总 RNA 的含量和质量，并采用 Affymetrix 微阵列系统进行 cDNA 表达谱测定。通过实时定量聚合酶链反应进一步验证微阵列分析结果。
分光光度分析显示，每位参与者纯化的总 RNA 量范围为 0.92μg/500μL 至 62.85μg/500μL。研究中两组总 RNA 量的差异较大，牙周炎组平均为 10.21μg/500μL±12.71μg/500μL，对照组平均为 15.97μg/500μL±23.47μg/500μL。进一步分析发现，大多数研究对象（9 例牙周炎和 6 例对照组）的 RNA 纯度（基于 A/A 比值）均在下游分析的可接受范围内（2.0±0.1）。研究样本显示 23S（3800bp）和 16S（1500bp）细菌 rRNA 特征的 2 条特征带。对 4 个样本（P2、P6、H5 和 H9）进行了初步微阵列分析。4 个样本中存在的基因百分比不一致，检测到的基因约为 1.8%-18.7%。实时定量聚合酶链反应证实，从每个样本中纯化的总 RNA 主要为细菌 RNA（Uni 16S），人 mRNA 极少。
本研究表明，从牙周炎患者和对照者的唾液中可以分离出少量人 RNA。如果要将唾液转录组用于确定个体患者的易感性，需要进一步改进从唾液中提取人 mRNA 的方法。
