microRNAs are small non-coding RNA molecules that operate as negative regulators of gene expression to regulate various biological functions. As researchers discover novel microRNAs and identify the significance of these little regulatory components connected to a vast array of biological processes, the study of microRNA is expanding quickly. Exosomes are small vesicles that are secreted by cells. Due to their presence of biomolecules, particularly miRNAs and isomiRs, they have gained interest for their potential application in biomarker development. Exosomes are present in plasma and other bodily fluids. The sequencing of miRNAs extracted from exosomes has the potential to find new disease biomarkers; however, the low RNA content of exosomes prevents accurate analysis and quantification. Here, we will cover many procedures involved in developing an improved short RNA (sRNA) library preparation technique for next-generation sequencing (NGS) miRNA analysis from blood samples or exosomes. We want to cover four different steps:

1-Protocols for isolating and enriching exosomes. We want to provide multiple techniques for isolating exosomes, including ultracentrifugation and extraction kits from diverse sample types, including serum, cell culture supernatant, urine, saliva, and other fluids.

2-RNA isolation. To offer appropriate methods for the separation of high-quality RNA.

3-RNA library preparation and next-generation sequencing. Specialized procedures for minimizing contamination and increasing the proportion of useable exosomal RNA sequence readings.

4-RNA-Seq data alignment, processing, and analysis. mRNAs and miRNAs undergo abundance difference analysis, family analysis, comparative analysis, and conservative analysis.