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Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations
Published on: August 4, 2016
Targeted gene-expression analysis by genome-controlled reverse transcription-PCR
Jakob Stenman1, Annukka Paju, Oso Rissanen
1Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland. jakob.stenman@helsinki.fi
Clinical Chemistry
|September 23, 2006
Summary
This study introduces a novel quantitative PCR assay for gene expression analysis, improving accuracy and reproducibility in formalin-fixed paraffin-embedded tissues for cancer diagnostics.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Quantitative PCR assays are crucial for tumor classification but require enhanced diagnostic accuracy and reproducibility.
- Analysis of gene expression in formalin-fixed paraffin-embedded (FFPE) tissues is essential for clinical applications.
Purpose of the Study:
- To develop a novel reverse transcription-PCR (RT-PCR) based quantitative assay.
- To improve the diagnostic accuracy and reproducibility of gene expression analysis.
- To enable gene expression analysis in FFPE tissue samples.
Main Methods:
- Developed an RT-PCR assay modifying cDNA to increase melting temperature of short amplicons.
- Utilized in-tube homogeneous melting-curve analysis for quantification.
- Applied the assay to analyze 8 genes in colon carcinoma cell lines, endoscopic biopsies, and archival FFPE/frozen tissues.
Main Results:
- The assay demonstrated a detection limit of 100 cDNA copies and a dynamic range of 3 orders of magnitude for FFPE samples.
- The prototype assay showed improved reproducibility compared to the TaqMan assay (interassay CVs 5%-20% vs 7%-43%).
- Successful gene expression analysis was achieved in 96% of biopsy, 86% of FFPE, and 100% of frozen samples.
Conclusions:
- The new technology combines competitive PCR reproducibility with accurate quantitative detection via in-tube melting-curve analysis.
- Enables efficient mRNA profiling in samples with limited cell numbers or small tissue amounts.
- Facilitates gene expression analysis in archival FFPE tissues, crucial for retrospective studies.
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