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Updated: Jul 11, 2026
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Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations
Published on: August 4, 2016
Genome-controlled reverse transcriptase-polymerase chain reaction for targeted gene-expression analysis.
J Stenman1, J Räsänen, T Tenkanen
1Department of Surgery, HUCH, Jorvi Hospital, Espoo, Finland. jakob.stenman@hus.fi
Scandinavian Journal of Clinical and Laboratory Investigation
|November 15, 2006
Summary
This study introduces a novel reverse transcriptase-PCR method for reproducible gene-expression analysis in various tissue types, including challenging FFPE samples. The new assay offers improved sensitivity and accuracy for molecular diagnostics.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Gene-expression profiling is crucial for tumor classification.
- Current polymerase chain reaction (PCR)-based methods lack reproducibility for diagnostics and formalin-fixed paraffin-embedded (FFPE) samples.
- Improved quantitative techniques are needed for accurate gene expression analysis.
Purpose of the Study:
- To develop a novel reverse transcriptase-PCR-based technology for quantitative assessment of multiple mRNA transcripts.
- To enhance reproducibility and enable gene expression analysis in FFPE tissues.
- To provide a sensitive method for analyzing small tissue or cell samples.
Main Methods:
- Utilized a multiplexed sequence modifying cDNA synthesis reaction.
- Employed competitive co-amplification of cDNA with genomic DNA as an internal standard.
- Quantified amplicon levels using in-tube homogeneous melting curve analysis.
Main Results:
- Achieved a dynamic range of three orders of magnitude and a detection limit of 100 cDNA molecules.
- Demonstrated improved reproducibility (inter-assay CV 5-20%) compared to TaqMan assays (7-43%).
- Successfully performed gene-expression analysis in 100% of frozen samples, 86% of FFPE samples, and 96% of biopsies.
Conclusions:
- The developed technique enables accurate mRNA expression analysis in cultured cells and endoscopic biopsies.
- Sensitive analysis of FFPE tissue is feasible due to the use of short PCR amplicons.
- This method offers a reproducible and sensitive approach for gene expression profiling in various clinical samples.
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