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MicroRNA (miRNA) are short, regulatory RNA transcribed from introns (non-coding regions of a gene) or intergenic regions (stretches of DNA present between genes). Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself, forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA...

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Related Experiment Video

Updated: May 10, 2026

MicroRNA Amplification and Recognition through Locked-nucleic-acid In situ Hybridization as a Novel Detection and Quantification Method

MicroRNA Amplification and Recognition through Locked-nucleic-acid In situ Hybridization as a Novel Detection and Quantification Method

Published on: October 7, 2025

Quantification of microRNA expression with next-generation sequencing.

Seda Eminaga¹, Danos C Christodoulou, Francois Vigneault

¹Department of Genetics, Harvard Medical School, Boston, MA, USA.

Current Protocols in Molecular Biology

|July 4, 2013

Summary

Next-generation sequencing enables comprehensive microRNA (miRNA) expression profiling. Barcoding multiplexes miRNA libraries to reduce costs, with a protocol and computational pipeline provided for analysis.

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Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Published on: April 14, 2015

Area of Science:

Molecular Biology
Genomics
Bioinformatics

Background:

Next-generation sequencing (NGS) technologies have revolutionized the study of microRNA (miRNA) expression.
Efficient and cost-effective methods are crucial for comprehensive miRNA profiling.

Purpose of the Study:

To provide a detailed protocol for isolating miRNAs and constructing multiplexed miRNA libraries.
To describe a computational pipeline for analyzing sequencing data from multiplexed miRNA libraries.

Main Methods:

Step-by-step protocol for miRNA isolation and library construction.
Barcoding strategy for multiplexing miRNA libraries.
Development of a custom computational pipeline for Illumina sequencing data analysis.

Main Results:

Successful isolation of miRNAs and construction of high-quality multiplexed libraries.
Demonstration of cost reduction through library multiplexing.
Validation of the computational pipeline for accurate analysis of sequencing reads.

Conclusions:

The presented protocol enables efficient and cost-effective miRNA expression profiling using multiplexed libraries.
The custom computational pipeline facilitates robust analysis of multiplexed miRNA sequencing data.
This approach supports comprehensive and accessible miRNA research.