A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools.

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• MicroRNAs (miRNAs) regulate a significant portion of human gene expression.
• Dysregulation of miRNA expression is implicated in various disease states.
• Accurate quantification of miRNAs is crucial for biomarker discovery and therapeutic target identification.

Purpose of the Study:

• To provide a detailed, step-by-step protocol for accurate microRNA quantification.
• To integrate experimental procedures with bioinformatic analysis for comprehensive miRNA profiling.
• To enable researchers to reliably measure miRNA expression from biological samples.

Main Methods:

• Isolation of microRNAs from biological tissues.
• Ligation of microRNAs to adaptors for high-throughput sequencing.
• Amplification and library preparation for sequencing.
• Bioinformatic analysis including read alignment, quantification, normalization, and differential expression analysis.

Main Results:

• A versatile and robust workflow for microRNA quantification is established.
• The method covers the entire process from tissue extraction to differential expression analysis.
• The protocol facilitates accurate measurement of miRNA expression profiles.

Conclusions:

• The presented combined experimental and bioinformatic approach offers a reliable method for microRNA quantification.
• This workflow aids in elucidating disease mechanisms and identifying potential therapeutic targets and biomarkers.
• Researchers can effectively utilize this protocol for comprehensive microRNA expression profiling.