High-Yield RNA-Extraction Method for Saliva

Abstract

BACKGROUND The use of salivary diagnostics is increasing because of its noninvasiveness, ease of sampling, and the relatively low risk of contracting infectious organisms. Saliva has been used as a biological fluid to identify and validate RNA targets in head and neck cancer patients. The goal of this study was to develop a robust, easy, and cost-effective method for isolating high yields of total RNA from saliva for downstream expression studies. METHODS Oral whole saliva (200 μL) was collected from healthy controls (n = 6) and from patients with head and neck cancer (n = 8). The method developed in-house used QIAzol lysis reagent (Qiagen) to extract RNA from saliva (both cell-free supernatants and cell pellets), followed by isopropyl alcohol precipitation, cDNA synthesis, and real-time PCR analyses for the genes encoding β-actin (“housekeeping” gene) and histatin (a salivary gland–specific gene). RESULTS The in-house QIAzol lysis reagent produced a high yield of total RNA (0.89–7.1 μg) from saliva (cell-free saliva and cell pellet) after DNase treatment. The ratio of the absorbance measured at 260 nm to that at 280 nm ranged from 1.6 to 1.9. The commercial kit produced a 10-fold lower RNA yield. Using our method with the QIAzol lysis reagent, we were also able to isolate RNA from archived saliva samples that had been stored without RNase inhibitors at −80 °C for >2 years. CONCLUSIONS Our in-house QIAzol method is robust, is simple, provides RNA at high yields, and can be implemented to allow saliva transcriptomic studies to be translated into a clinical setting.