Detection of Serum microRNAs From Department of Defense Serum Repository: Correlation With Cotinine, Cytokine, and Polycyclic Aromatic Hydrocarbon Levels

Abstract

Objective: The aim of this study was to investigate whether serum samples from the Department of Defense Serum Repository (DoDSR) are of sufficient quality to detect microRNAs (miRNAs), cytokines, immunoglobulin E (IgE), and polycyclic aromatic hydrocarbons (PAHs).

Methods: MiRNAs were isolated and quantified by polymerase chain reaction (PCR) array. Cytokines and chemokines related to inflammation were measured using multiplex immunoassays. Cotinine and IgE were detected by enzyme-linked immunoassay (ELISA) and PAHs were detected by Liquid Chromatography/Mass Spectroscopy.

Results: We detected miRNAs, cytokines, IgE, and PAHs with high sensitivity. Eleven of 30 samples tested positive for cotinine suggesting tobacco exposure. Significant associations between serum cotinine, cytokine, IgE, PAHs, and miRNA were discovered.

Conclusion: We successfully quantified over 200 potential biomarkers of occupational exposure from DoDSR samples. The stored serum samples were not affected by hemolysis and represent a powerful tool for biomarker discovery and analysis in retrospective studies.

FIGURE 1

Isolation and detection of miRNAs from serum. Schematic outline of serum miRNA isolation, quality control, and profiling analysis. Briefly, 150 μL of serum from DoDSR samples was used to isolate miRNA using affinity column based chemistry. After isolation, a small portion of sample was used in single miRNA RT-qPCR assays to demonstrate successful miRNA analysis. Following successful single qPCR reactions, samples were profiled using qPCR assays for over 150 miRNAs in duplicate in a 384-well plate. Data were then processed and normalized to determine relative expression levels of each miRNA in the serum samples.

FIGURE 2

Identification and quality assessment of miRNAs present in serum. (A), 30 archival samples are listed on the x-axis with the number of miRNAs detected in each sample on the left side y-axis. The average PCR threshold cycle (Ct) for detected miRNA per sample is listed in the right side y axis. (B) Summary of miRNA profiling data. One hundred seventy-seven miRNAs were assayed with an average of 111 miRNAs detected per sample. (C) Assessment of red blood cell lysis (hemolysis) by detection of two different miRNAs. MiR-451 is present in red blood cells, while miR-23a is not. The difference in Ct (dCt) value between miR-23a and miR-451 can be used to assess hemolysis. Samples with a dCt value 7.0 or greater would be flagged for potential hemolysis (any sample with dCt above dotted line). All DoDSR samples had a dCt value of 5.5 or lower, demonstrating that hemolysis is not a concern.

FIGURE 3

Pathway analysis of miRNAs differentially expressed in cotinine-positive samples. Pathway analysis (see Materials and Methods) of differentially expressed miR-NAs in cotinine-positive samples based on ranking according to P values was performed in R (R package miRNApath for pathway analysis). Serum cotinine levels were used to group the samples into tobacco users (n =11) compared with non-tobacco users (n =19) as described in the Materials and Methods section. The color map P values indicate the strength of the association between the miRNA and the relevant pathway. Pathways targeted by fewer than two miRNAs and miRNAs that targeted fewer than two pathways were omitted for clarity.

FIGURE 4

Individual Spearman rank correlations between specific serum components, including cytokines, miRNAs, and IgE. Different serum components were analyzed for correlation using Spearman rank method. Each correlation used all 30 DoDSR samples and these analyses were done independently of cotinine status. (A) IL-1β and IL-6 levels are highly correlated in DoDSR serum samples. (B) The serum expression of miR-423-5p correlates with IL-10 levels. (C) There is a negative correlation between let-7b-5p and Tumor necrosis factorα levels. (D) Serum IgE levels correlate with expression of miR-22-3p.

FIGURE 5

Cluster analysis of correlations between miRNA and cytokines. The miRNAs (y-axis) with significant P values for >3 cytokines (x-axis) are plotted. The complete set of correlations is provided as supplemental table 1 (http://links.lww.com/JOM/A269). The color map from yellow to red indicate lower to high –log10 (P values).

FIGURE 6

Spearman rank correlation between miR-142-5p and anthracene. Significant correlations between PAH levels and specific miRNAs were identified. (A) Serum anthracene levels and miR-148b-3p expression were analyzed for correlation by Spearman rank method. (B) Serum naphthalene levels and miR-19b-3p expression were analyzed for correlation by Spearman rank method.