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RNA-seq

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based...

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Related Experiment Video

Updated: May 23, 2026

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

Published on: August 3, 2011

High-throughput multiplex sequencing of miRNA.

Francois Vigneault^1,2,3, Dmitry Ter-Ovanesyan^2,4, Shahar Alon^5,6

¹Department of Genetics, Harvard Medical School, Boston, Massachusetts.

Current Protocols in Human Genetics

|April 4, 2012

Summary

Multiplexing microRNA libraries using PCR bar-coding for next-generation sequencing offers cost advantages. This method avoids bias introduced by ligation bar-coding and prevents adapter dimer formation, enabling efficient miRNA expression profiling.

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Last Updated: May 23, 2026

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

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Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

Published on: November 18, 2014

Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

Next-generation sequencing (NGS) provides superior microRNA (miRNA) expression profiling capabilities, including higher sample throughput and novel miRNA discovery.
Multiplexing multiple samples in a single NGS run significantly reduces costs, as current sequencing depths often exceed quantification needs for miRNAs.
Previous bar-coding strategies at the ligation step introduced bias in miRNA expression data, compromising accuracy.

Purpose of the Study:

To develop and present a user-friendly PCR bar-coding method for preparing multiplexed miRNA libraries for Illumina sequencing.
To provide an alternative bar-coding approach that mitigates bias associated with ligation-based methods.
To optimize the library preparation workflow for efficiency and accuracy in miRNA expression profiling.

Main Methods:

A novel PCR bar-coding strategy was implemented for preparing miRNA libraries.
The method was designed for compatibility with Illumina sequencing platforms.
The protocol was streamlined for a one-day completion time and to prevent adapter dimer formation.

Main Results:

The developed PCR bar-coding method successfully enabled multiplexing of miRNA libraries.
This approach effectively avoided the significant bias previously observed with ligation-based bar-coding.
The method also demonstrated efficacy in preventing the formation of adapter dimers, a common artifact.

Conclusions:

PCR bar-coding represents a reliable and unbiased strategy for multiplexing miRNA libraries for NGS.
This user-friendly method offers a cost-effective and efficient solution for accurate miRNA expression profiling.
The protocol facilitates high-throughput analysis and discovery of miRNA expression patterns.