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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.
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Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific...

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Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
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Related Experiment Video

Updated: Jun 24, 2025

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

Linearly Amplified Single-Stranded RNA-Derived Transcriptome Sequencing (LAST-seq).

Jun Lyu¹, Chongyi Chen¹

¹Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

|June 14, 2024

Summary

This summary is machine-generated.

We developed LAST-seq, a new single-cell RNA sequencing method. It directly amplifies RNA, improving sensitivity and reducing noise for better gene expression analysis.

Keywords:

Gene expression noise In vitro transcription Linear amplification Single-cell RNA sequencing Single-stranded RNA template Transcriptional bursting

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Related Experiment Videos

Last Updated: Jun 24, 2025

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

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

Published on: August 4, 2016

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:

Single-cell RNA sequencing (scRNA-seq) is crucial for biological and biomedical research.
Current scRNA-seq methods use reverse transcription (RT) and second-strand synthesis (SSS), often limiting detection sensitivity.
Inefficiencies in RT/SSS compromise the accurate quantification of RNA in single cells.

Purpose of the Study:

To develop a novel scRNA-seq method with enhanced sensitivity and reduced technical noise.
To overcome the limitations of traditional RT/SSS-dependent scRNA-seq protocols.
To enable more precise analysis of gene expression at the single-cell level.

Main Methods:

Developed linearly amplified single-stranded RNA-derived transcriptome sequencing (LAST-seq).
LAST-seq directly amplifies single-stranded RNA, bypassing the need for RT and SSS.
Utilized an RNase H/DNA polymerase strategy to attach a T7 promoter to single-stranded RNA, followed by T7 promoter-mediated in vitro transcription (IVT).

Main Results:

LAST-seq demonstrated high-sensitivity RNA detection in single-cell transcriptome analysis.
The method achieved a low level of technical noise compared to existing techniques.
Successfully applied LAST-seq to quantify transcriptional bursting kinetics in human cells.

Conclusions:

LAST-seq offers a significant advancement in single-cell RNA sequencing technology.
The method provides a more sensitive and less noisy approach for transcriptome analysis.
LAST-seq facilitates deeper insights into gene expression regulation, including the role of chromatin organization.