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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...

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Updated: May 6, 2026

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

Published on: October 9, 2020

Small RNA library construction from minute biological samples.

Jessica A Matts¹, Yuliya Sytnikova, Gung-Wei Chirn

¹Department of Biology, Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, MA, USA.

Methods in Molecular Biology (Clifton, N.J.)

|November 2, 2013

Summary

This study presents improved methods for constructing small RNA cDNA libraries from minute tissue samples. These techniques enhance the characterization of regulatory small RNAs like microRNAs (miRNAs) using deep sequencing.

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Last Updated: May 6, 2026

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

Published on: October 9, 2020

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Published on: October 27, 2011

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

Published on: July 2, 2016

Area of Science:

Molecular Biology
Genomics
Biochemistry

Background:

Deep sequencing is crucial for discovering small regulatory RNAs (miRNAs, endo-siRNAs, piRNAs).
Constructing cDNA libraries from minute tissue samples is challenging due to dominant structural RNA fragments.
Ribosomal RNA (rRNA) fragments are common contaminants in small RNA preparations from limited tissues.

Purpose of the Study:

To describe methods for successful small RNA library construction from minute tissues.
To address challenges in identifying regulatory small RNAs in limited sample amounts.
To improve the characterization of miRNAs, endo-siRNAs, and piRNAs.

Main Methods:

Isolation of small RNAs from minute Drosophila ovarium follicle cell tissues.
Application of antisense oligonucleotide depletion to remove contaminating rRNA.
Utilizing an acryloylaminophenylboronic acid (APB) polyacrylamide gel system for RNA separation.
Generation of cDNA libraries compatible with high-throughput sequencing platforms.

Main Results:

Demonstrated effective depletion of abundant 2S rRNA from small RNA preparations.
Successfully separated endo-siRNAs and piRNAs from rRNA contaminants.
Generated small RNA libraries suitable for multiplex sequencing on the Illumina Hi-Seq platform.

Conclusions:

The described methods significantly improve small RNA library construction from minute tissue samples.
These techniques enhance the accurate identification and characterization of regulatory small RNAs.
The methodology facilitates deep-sequencing applications for small RNA discovery in limited biological material.