MicroRNA biomarker discovery in depressed patients using the NanoString nCounter technology

Background
Major depressive disorder (MDD) is the leading cause of disability worldwide according to the World Health Organization. In Denmark, depression affects approximately 3–7% of the population at any given time, with a lifetime prevalence of 18%. Although the 2024 Danish Depression Database reports modest improvements in disease monitoring and treatment, major clinical challenges persist, particularly in early diagnosis and individualized treatment selection. In the psychiatric field, the diagnosis is made through clinical examination and based only on symptoms. This is problematic as MDD symptoms are partly overlapping with other mental disorders. Furthermore, sex differences in prevalence, symptoms, and responses to treatment also exist, adding to the complexity of MDD1. Antidepressant response is highly variable, with many patients experiencing delayed clinical improvement or treatment resistance, and therapeutic efficacy observed in only up to 60% of cases after several weeks. These limitations underscore the need for clinically accessible biomarkers to support diagnosis, guide treatment stratification, and enable earlier assessment of therapeutic response.

Today, it is generally accepted that the etiology of MDD is multifactorial with multiple susceptibility genes2 interacting with environmental factors. These factors may contribute partly through epigenetic processes, regulating gene expression at the transcriptional and post-transcriptional level by non-coding RNAs (ncRNAs)3,4. Recently, it has been reported that ncRNAs can influence the development of MDD, yet microRNAs (miRNAs) are the best studied ncRNAs in terms of functionality and relevance to psychiatric disorders5-7. Clinically, miRNAs offer great potential as biomarkers. In blood the dysregulation of miRNAs has been intensively studied in relation to MDD and treatment thereof 5,8-10. Despite substantial effort, the results are still non-conclusive.

Aims & Objectives
We hypothesize that sex, different blood samples, miRNA extraction protocols, and detection methods, as well as extracellular vesicles (EVs) may influence the miRNA results. In the given study, we will use the NanoString nCounter technology and specifically focus on the RNA extraction and EVs in plasma samples from the Cimbi Biobank11.

Method
Samples: Age- and sex-matched plasma samples from depressed patients, taken before and after 8 weeks treatment with escitalopram and from healthy controls are included in the study. The samples were taken as part of the Neuropharm project11.

miRNA extraction: In plasma samples we have tested three different kits for RNA extraction.

miRNA profiling: We will use the NanoString nCounter technology and the Hu v3 miRNA codeset kit with 827 miRNAs for profiling of the plasma samples.

Results
miRNA extraction: We find that the Norgen Plasma/Serum Circulating and Exosomal RNA Purification Kit, Slurry Format perform superior. Different input and elution volumes were tested to find the optimal settings in combination with the NanoString nCounter technology.

The miRNA profiling is ongoing.

Discussion & Conclusions
Our preliminary miRNA data with this Danish cohort demonstrates sex differences, and miRNA signatures associated with MDD and antidepressant treatment response. The findings support the clinical utility of circulating miRNAs as biomarkers for precision psychiatry and may facilitate more targeted and timely treatment strategies for patients with MDD in the future.