Park Noh Jin, Li Yang, Yu Tianwei, Brinkman Brigitta M N, Wong David T
Dental Research Institute, UCLA School of Dentistry, Johnson Comprehensive Cancer Center, Division of Head and Neck Surgery/Otolaryngology, Henry Samueli School of Engineering, UCLA, Los Angeles, CA 90095, USA.
Clin Chem. 2006 Jun;52(6):988-94. doi: 10.1373/clinchem.2005.063206. Epub 2006 Apr 6.
We have previously shown that human mRNAs are present in saliva and can be used as biomarkers of oral cancer. In this study, we analyzed the integrity, sources, and stability of salivary RNA.
We measured the integrity of salivary RNA with reverse transcription followed by PCR (RT-PCR) or RT-quantitative PCR (RT-qPCR). To study RNA entry sites into the oral cavity, we used RT-PCR analysis of salivary RNA from the 3 major salivary glands, gingival crevice fluid, and desquamated oral epithelial cells. We measured stability of the salivary beta-actin mRNA by RT-qPCR of salivary RNA incubated at room temperature for different periods of time. We measured RNA association with other macromolecules by filtering saliva through pores of different sizes before performing RT-qPCR. To assess RNA-macromolecule interaction, we incubated saliva with Triton X-100 for different periods of time before performing RT-qPCR.
In most cases, we detected partial- to full-length salivary mRNAs and smaller amounts of middle and 3' gene amplicons compared with the 5'. RNA was present in all oral fluids examined. Endogenous salivary beta-actin mRNA degraded more slowly than exogenous beta-actin mRNA, with half-lives of 12.2 and 0.4 min, respectively (P <0.001). Salivary RNA could not pass through 0.22 or 0.45 mum pores. Incubation of saliva with Triton X-100 accelerated degradation of salivary RNA.
Saliva harbors both full-length and partially degraded forms of mRNA. RNA enters the oral cavity from different sources, and association with macromolecules may protect salivary RNA from degradation.
我们之前已经表明，人类mRNA存在于唾液中，并且可以用作口腔癌的生物标志物。在本研究中，我们分析了唾液RNA的完整性、来源和稳定性。
我们通过逆转录随后进行PCR（RT-PCR）或RT定量PCR（RT-qPCR）来测量唾液RNA的完整性。为了研究RNA进入口腔的位点，我们对来自3个主要唾液腺、龈沟液和脱落口腔上皮细胞的唾液RNA进行了RT-PCR分析。我们通过对在室温下孵育不同时间段的唾液RNA进行RT-qPCR来测量唾液β-肌动蛋白mRNA的稳定性。在进行RT-qPCR之前，我们通过用不同大小孔径的滤膜过滤唾液来测量RNA与其他大分子的结合。为了评估RNA-大分子相互作用，在进行RT-qPCR之前，我们将唾液与Triton X-100孵育不同时间段。
在大多数情况下，与5'端相比，我们检测到部分至全长的唾液mRNA以及较少量的中间和3'基因扩增子。在所检测的所有口腔液体中均存在RNA。内源性唾液β-肌动蛋白mRNA的降解速度比外源性β-肌动蛋白mRNA慢，半衰期分别为12.2分钟和0.4分钟（P<0.001）。唾液RNA无法通过0.22或0.45μm的孔径。唾液与Triton X-100孵育会加速唾液RNA的降解。
唾液中含有全长和部分降解形式的mRNA。RNA从不同来源进入口腔，并且与大分子的结合可能保护唾液RNA不被降解。
