Assessing the impact of storage conditions on RNA from human saliva and its application to the identification of mRNA biomarkers for asthma

Abstract

Introduction: Human saliva was used to develop non-invasive liquid biopsy biomarkers to establish saliva as an alternate to blood and plasma in translational research. The present study focused on understanding the impact of sample storage conditions on the extraction of RNA from saliva and the RNA yield, to be applied in clinical diagnosis. In this study, genes related to asthma were used to test the method developed. Methods: Salivary RNA was extracted from three subjects using the Qiazol^® based method and quantified by both spectrophotometric (NanoDrop) and fluorometric (Qubit^®) methods. RNA integrity was measured using a bioanalyzer. Quantitative PCR was used to monitor the impact of storage conditions on the expression of housekeeping genes: GAPDH and β-actin, and the asthma related genes: POSTN and FBN2. In addition, an independent cohort of 38 asthmatics and 10 healthy controls were used to validate the expression of POSTN and FBN2 as mRNA salivary biomarkers. Results: Approximately 2 µg of total RNA was obtained from the saliva stored at 40°C without any preservative for 2 weeks showing consistent gene expression with RNA stored at room temperature (RT) for 48 h with RNAlater. Although saliva stored with RNAlater showed a substantial increase in the yield (110 to 234 ng/μL), a similar Cq (15.6 ± 1.4) for the 18s rRNA gene from saliva without preservative showed that the RNA was stable enough. Gene expression analysis from the degraded RNA can be performed by designing the assay using a smaller fragment size spanning a single exon as described below in the case of the POSTN and FBN2 genes in the asthma cohort. Conclusion: This study showed that samples stored at room temperature up to a temperature of 40°C without any preservative for 2 weeks yielded relatively stable RNA. The methodology developed can be employed to transport samples from the point of collection to the laboratory, under non-stringent storage conditions enabling the execution of gene expression studies in a cost effective and efficient manner.

Keywords: RNA extraction; degradation; diagnosis; gene expression; salivary RNA.

FIGURE 1

Schematic for the saliva sample processing to test the effect of storage conditions (temperature and duration) on RNA extraction. The initial sample collected from three healthy controls was distributed into different time points (48 h and 2 weeks) which were further distributed into temperatures −80°C, RT, and 40°C. The sample at each temperature was stored either with or without the preservative. All the samples were processed for extraction of RNA at the end of the time point.

FIGURE 2

Effect of the storage conditions on RNA yield and integrity. Sample 1 through Sample 12 (Supplementary Table S4) is the Turbo DNase-treated RNA sample obtained from the saliva samples stored at RT, −80°C, and 40°C for 48 h and 2 weeks with/without RNAlater. (A) Graph showing the RNA quantity obtained from the saliva samples stored at different temperatures and time points with/without the RNA stabilizing agent. ** signifies p < 0.01; ns, not significant. The details on the output from statistical analysis are provided in Supplementary Table S2. (B) Representative electropherogram obtained from Agilent Bioanalyzer for the RNA samples obtained from the saliva samples stored at different temperatures and time points with/without RNAlater. The ladder size was mentioned as the number of the nucleotides. Sample ID and their corresponding description is provided in Supplementary Table S3. The bands corresponding to 28S rRNA and 18S rRNA were marked as 28S and 18S, respectively. The red dashes above the electropherogram indicate flags from the instrument and are displayed when the input RNA does not fit the standard total RNA profile.

FIGURE 3

Expression analysis for housekeeping genes across the storage conditions among the three subjects. Bar plots for ΔCq values for housekeeping genes A and B. ACTB and C and D. GAPDH with variable amplicon sizes spanning single and double exons as shown in Table 1. ACTB1 (A) and ACTB2 (B) are less than 120-bp amplicons spanning two and single exons, respectively. GAPDH1 (C) and GAPDH2 (D) are less than 150-bp amplicons spanning two and single exons, respectively. In all the cases, there was no significant change noticed across the storage conditions.

FIGURE 4

Differential gene expression for the genes related to asthma among 20 non-severe and 18 severe asthma patients’ saliva as compared to 10 healthy controls. (A) FBN2 and (B) POSTN. The relative expression compared to healthy controls was presented as fold change (2^−ΔΔCq). The Mann–Whitney test was conducted to test the significance between the groups. p < 0.05 was considered statistically significant.