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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 5, 2026

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

Targeted RNA-sequencing with competitive multiplex-PCR amplicon libraries.

Thomas M Blomquist¹, Erin L Crawford, Jennie L Lovett

¹Department of Medicine, University of Toledo Health Sciences Campus, Toledo, Ohio, United States of America.

|November 16, 2013

Summary

A new competitive multiplex PCR method enables accurate and reproducible targeted RNA-sequencing for molecular diagnostics. This approach significantly reduces sequencing reads, making it cost-effective and suitable for clinical applications.

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Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

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Published on: February 2, 2024

Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

Whole transcriptome RNA-sequencing is powerful but costly and data-intensive for diagnostics.
Existing methods face challenges in reproducibility and cost-effectiveness for routine molecular testing.

Purpose of the Study:

To develop a targeted quantitative RNA-sequencing method suitable for molecular diagnostics.
To improve reproducibility and reduce sequencing read requirements for transcript quantification.

Main Methods:

Developed a competitive multiplex PCR-based amplicon sequencing library preparation method.
Incorporated synthetic competitive template internal standards to control for PCR amplification variation.
Utilized PCR conditions to normalize amplicon concentrations for equimolar output.

Main Results:

Demonstrated excellent reproducibility (R² = 0.997) and 97% accuracy for 2-fold change detection using ERCC materials.
Achieved high concordance across inter-day, inter-site, and inter-library preparations (R² = 0.97-0.99) with SEQC materials.
Showcased cross-platform concordance with TaqMan qPCR (R² = 0.96) and traditional Illumina RNA-sequencing (R² = 0.94).
Reduced sequencing reads by over 10,000-fold for quantifying over 100 transcripts across a 10⁷-fold expression range.

Conclusions:

The competitive multiplex PCR method offers quality control and reproducibility for targeted RNA-sequencing.
This technique significantly reduces sequencing needs, facilitating the development of RNA-sequencing biomarkers for molecular diagnostics.