RNAPro•SAL: a device for rapid and standardized collection of saliva RNA and proteins

Abstract

The stabilization and processing of salivary transcriptome and proteome biomarkers is a critical challenge due to the ubiquitous nature of nucleases and proteases as well as the inherent instability of these biomarkers. Furthermore, extension of salivary transcriptome and proteome analysis to point-of-care and remote sites requires the availability of self-administered ambient temperature collection and storage tools. To address these challenges, a self-contained whole saliva collection and extraction system, RNAPro•SAL, has been developed that provides rapid ambient temperature collection along with concurrent processing and stabilization of extracellular RNA (exRNA) and proteins. The system was compared to the University of California, Los Angeles (UCLA) standard clinical collection process (standard operating procedure, SOP). Both systems measured total RNA and protein, and exRNA IL-8, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin and ribosomal protein S9 (RPS9) by qPCR. Proteome analysis was measured by EIA analysis of interleukin-8 (IL-8), and β-actin, as well as total protein. Over 97% of viable cells were removed by both methods. The system compared favorably to the labor-intensive clinical SOP, which requires low-temperature collection and isolation, yielding samples with similar protein and exRNA recovery and stability.

Keywords: ambient temperature; point-of-care settings; saliva diagnostic tool; salivary extracellular RNA; salivary protein.

Figure 1. The RNAPro•SAL system and scheme for experimental validation

(A) Diagram of RNAPro•SAL system. (B) Experimental design. A 10 mL whole saliva (WS) sample (tube A) was collected on ice (4°C) and split into 2 5 mL subsamples (tubes B & C). Saliva supernatant (SS) (tube D) was prepared from tube B using the standard clinical collection process (SOP) by centrifugation at 2600 × g for 15 min at 4°C. Stabilizers were added as described in the text and supplemental data. SS samples obtained via SOP were split from tube D into four aliquots (tubes D1–D4) for independent storage and time point analysis. RNAPro•SAL SS (tube E) was prepared by flowing the WS sample in tube C through the RNAPro•SAL bifurcated filter. Collection tubes were combined. The SS samples obtained via RNAPro•SAL were split from tube E into four aliquots (tubes E1–E4) for independent storage and time point analysis.

Figure 2. Comparison of total RNA (A), protein recovery (B), and cell removal (C) for salivary specimens

Samples were obtained using the standard operating procedure with storage at room temperature (SOP) and the RNAPro•SAL system.

Figure 3. Comparison of transcriptomic and proteomic recovery at ambient temperature as a function of time for saliva specimens

Amplifiable transcript (RNA) levels were assessed by RT-qPCR and reported as mean C_q for 8 participants run in duplicate at each time point (n = 16). Standard operating procedure (SOP) and RNAPro•SAL stored at ambient temperatures: (A) GAPDH, (B) β-actin, (C) ribosomal protein S9 (RPS9), and (D) interleukin-8 (IL-8). RNAPro•SAL ± SUPERase-IN stored at ambient temperature and RNAPro•SAL at 4°C (E) GAPDH, (F) β-actin, (G) RPS9, and (H) IL-8. (I) Total protein recovery in µg/mL was determined by bicinchoninic acid (BCA) protein assay. (J) Specific protein recovery for IL-8 and β-actin was determined using ELISA and reported as the index of the log ratio of RNAPro•SAL/SOP with respect to time. RNAPro•SAL protein levels were adjusted for 20% ethanol dilution.