High-Quality and High-Yield RNA Extraction Method From Whole Human Saliva.

Abstract

Background: Human saliva has been identified as a novel, practical, and noninvasive source of biomarkers and genetic materials. However, it is equally challenging due to the availability of an abundance of impurities in the form of microbes and other proteinaceous compounds. The objective of this study was to develop a robust, reproducible, and economic method of extracting high-yield and high-quality RNA from whole human saliva.

Methods: The modified TRIzol protocol was developed to extract RNA from saliva (n = 14), followed by complementary DNA synthesis and reverse transcription quantitative polymerase chain reaction analyses for the genes encoding IL1B, ALPL, RUNX2, and ACTB. To compare our protocol with the spin column-based method, we used Qiagen Salivary Protect Micro-RNA spin columns (n = 6). To evaluate and compare the yields and quality of extracted RNAs from both methods, we used (1) Experion Bioanalyzer, (2) QuantiFluor RNA dye, and (3) NanoDrop 2000 Spectrometer.

Results: With the modified TRIzol lysis protocol, a high yield of total RNA, on average 12.34 μg, from saliva was extracted compared with on average 0.2 μg with a spin column-based method. The average RQI (RNA quality index) with the TRIzol method was 7.86, which is also comparable with that of the spin column-based method (RQI = 7.58). QuantiFluor dye used for RNA quantification showed a 16-fold higher yield of RNA concentration using our TRIzol protocol.

Conclusions: Our modified TRIzol protocol is a reproducible method to extract RNA from whole human saliva which can be used for gene expression analysis. This method allows also ensures the quality of RNA required for specific applications such as RNA sequencing.