Confounding Factors Impacting microRNA Expression in Human Saliva: Methodological and Biological Considerations

Abstract

There is growing interest in saliva microRNAs (miRNAs) as non-invasive biomarkers for human disease. Such an approach requires understanding how differences in experimental design affect miRNA expression. Variations in technical methodologies, coupled with inter-individual variability may reduce study reproducibility and generalizability. Another barrier facing salivary miRNA biomarker research is a lack of recognized “control miRNAs”. In one of the largest studies of human salivary miRNA to date (922 healthy individuals), we utilized 1225 saliva samples to quantify variability in miRNA expression resulting from aligner selection (Bowtie1 vs. Bowtie2), saliva collection method (expectorated vs. swabbed), RNA stabilizer (presence vs. absence), and individual biological factors (sex, age, body mass index, exercise, caloric intake). Differential expression analyses revealed that absence of RNA stabilizer introduced the greatest variability, followed by differences in methods of collection and aligner. Biological factors generally affected a smaller number of miRNAs. We also reported coefficients of variations for 643 miRNAs consistently present in saliva, highlighting several salivary miRNAs to serve as reference genes. Thus, the results of this analysis can be used by researchers to optimize parameters of salivary miRNA measurement, exclude miRNAs confounded by numerous biologic factors, and identify appropriate miRNA controls.

Exosomal miRNAs as Biomarkers of Ischemic Stroke

“…Another gap facing the field of exosomal miRNA biomarker discovery is related to the potential confounding factors affecting miRNA expression. Indeed, there is a considerable body of the literature on how miRNA expression may depend on individual factors, such as sex, age, body mass index, diet, exercise, and comorbidities, which could influence findings [92][93][94][95][96][97][98]. However, most studies did not consider the potential influence of such factors on exosomal miRNA expression.…”

Review on the Role of Salivary Biomarkers in the Diagnosis of Mild Traumatic Brain Injury and Post-Concussion Syndrome

“…MicroRNAs (miRNAs) are small, non-coding RNA molecules that play essential roles in messenger RNA (mRNA) translation. MiRNAs epigenetically repress mRNA translation, based on complementary binding to their sequences, promoting mRNA degradation [ 44 ]. There are various molecular pathways in which miRNA can be detected in peripheral fluids following TBI.…”

“…Recently, saliva has emerged as a valuable source of miRNAs for diagnostic purposes, especially in some oral or esophageal cancers [ 48 , 49 ]. Saliva contains a wide range of miRNAs that are stable and easily accessible, making it a non-invasive source of biological information, in some cases regarding complete signaling pathways [ 44 ].…”

The Role of MicroRNAs in Breast Cancer and the Challenges of Their Clinical Application

“…On the other hand, miRNA levels can be influenced by several factors, including age, gender, and the presence of disease, further complicating their use as reliable markers [164]. Another concern arises from the fact that certain potential miRNA biomarkers are ubiquitously present in both healthy individuals and those with cancer [165].…”