An optimised saliva collection method to produce high-yield, high-quality RNA for translational research

Abstract

Saliva represents an ideal matrix for diagnostic biomarker development as it is readily available and requires no invasive collection procedures. However, salivary RNA is labile and rapidly degrades. Previous attempts to isolate RNA from saliva have yielded poor quality and low concentrations. Here we compare collection and processing methods and propose an approach for future studies. The effects of RNA stabilisers, storage temperatures, length of storage and fasting windows were investigated on pooled saliva samples from healthy volunteers. Isolated RNA was assessed for concentration and quality. Bacterial growth was investigated through RT-PCR using bacterial and human primers. Optimal conditions were implemented and quality controlled in a clinical setting. The addition of RNAlater increased mean RNA yield from 4912 ng/μl to 15,473 ng and RNA Integrity Number (RIN) from 4.5 to 7.0. No significant changes to RNA yield were observed for storage at room temperature beyond 1 day or at -80˚C. Bacterial growth did not occur in samples stored at ambient temperature for up to a week. There was a trend towards higher RNA concentration when saliva was collected after overnight fasting but no effect on RIN. In the clinic, RNA yields of 6307 ng and RINs of 3.9 were achieved, improving on previous reports. The method we describe here is a robust, clinically feasible saliva collection method using preservative that gives high concentrations and improved RINs compared to saliva collected without preservative.

Performance of Severe Acute Respiratory Syndrome Coronavirus 2 Real-Time RT-PCR Tests on Oral Rinses and Saliva Samples

“…One of the main concerns related to saliva collection has been the stability of RNA in saliva samples, which has been extensively researched over the years 10 . Samples were collected in 50-mL conical tubes that are readily available and inexpensive and SARS-CoV-2 RNA viral load remained stable for up to 24 hours at room temperature or refrigerated, well within expected time to receipt in the laboratory (less than 24 hours) at MSK.…”

Loop‐mediated isothermal amplification for the detection of SARS‐CoV‐2 in saliva

“…Indeed, the addition of the RNase inhibitor improved the sensitivity of the RT‐LAMP assay on spiked saliva samples. This indicates that the RNase activity of saliva is one of the major issues in salivary RNA diagnostics (Fábryová and Celec, 2014; Sullivan et al ., 2020).…”

SYBR green one-step qRT-PCR for the detection of SARS-CoV-2 RNA in saliva

“…The reduced sensitivity is likely the result of: (I) increased spontaneous RNA cleavage at higher temperatures, and (II) enzymatic degradation by salivary ribonucleases post-heat treatment, which may also denature protective proteins or RNA secondary structures (Brisco and Morley 2012;Emilsson et al 2003) . This degradation may be circumvented by the addition of RNA stabilisers, such as 6-8% formamide (Yasukawa et al 2010) , prior to heat treatment or with an alternate buffer with greater pH stability at higher temperatures (Sullivan et al 2020;Reineke et al 2011) . However, caution is required as buffer composition drastically affects RT-PCR performance as highlighted by no amplification with PBS, although it has been shown to be a suitable medium for transporting samples (Rodino et al 2020) .…”

“…It may also be possible to apply ultraviolet radiation to saliva samples or temporarily alter the pH, however, it may be challenging to find a treatment leading to viral inactivation without complete RNA damage (Darnell et al 2004;Lemire et al 2016;Beck et al 2015) . Alternatively, RNA extractions could be performed on saliva for which clinically-relevant methods now exist, however, this would still add considerable costs and labour (Pandit et al 2013;Sullivan et al 2020) . Methods for one-step RNA extractions may provide a compromise between direct qRT-PCR and complete RNA extraction (Sentmanat et al 2020) .…”