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Clinical Chemistry 52: 1988-1996, 2006. First published September 21, 2006; 10.1373/clinchem.2006.073064
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(Clinical Chemistry. 2006;52:1988-1996.)
© 2006 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Targeted Gene-Expression Analysis by Genome-Controlled Reverse Transcription-PCR

Jakob Stenman1,2,a,3, Annukka Paju3,3, Oso Rissanen3, Tuomas Tenkanen4,3, Caj Haglund5,6, Jari Räsänen7, Jarmo Salo7, Ulf-Håkan Stenman3,8 and Arto Orpana3,8,9

1 Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland.
2 Department of Surgery, HUCH, Jorvi Hospital, Espoo, Finland.
3 Departments of Clinical Chemistry,5 Surgery,6 Pathology, and9 Medical Genetics, University of Helsinki, Helsinki, Finland.
4 Finnzymes Oy, Espoo, Finland.
7 Division of General Thoracic and Esophageal Surgery, Department of Cardiothoracic Surgery, HUCH, Helsinki, Finland.
8 HUCH Laboratory Diagnostics, Helsinki, Finland.

aAddress correspondence to this author at: Hospital for Children and Adolescents, University of Helsinki, P.O. Box 281, FIN 00029 HUS, Finland. Fax 358-9-4717-1731; e-mail jakob.stenman{at}helsinki.fi.

Background: For gene-expression analysis, which is anticipated to play an important role in classification of tumors and premalignant conditions, PCR-based quantitative assays must have increased diagnostic quantitative accuracy and reproducibility and enable analysis of gene expression in formalin-fixed paraffin-embedded (FFPE) tissue samples.

Methods: We developed a reverse transcription–PCR-based quantitative assay that modifies the cDNA sequence to increase the melting temperature of short (56–64 bp) PCR amplicons, enabling their quantification in-tube by homogeneous melting-curve analysis. We used this method to analyze the expression of 8 genes, 7 potential colon cancer markers, and 1 control in samples obtained from 3 colon carcinoma cell lines, endoscopic biopsy from 8 patients undergoing gastroscopy for Barrett esophagus, and archival FFPE and frozen tissue from 20 patients who underwent surgery for colon carcinoma.

Results: The detection limit of the assay, when optimized for FFPE samples, was 100 copies of cDNA, and the dynamic range was 3 orders of magnitude. A prototype assay containing a panel of 8 genes displayed good reproducibility compared with the commercially available TaqMan® assay (interassay CVs, 5%–20% vs 7%–43%, respectively). Gene-expression analysis was performed successfully in 26 (96%) of 27 endoscopic biopsy specimens, 30 (86%) of 35 archival FFPE samples, and 20 (100%) of 20 archival frozen samples.

Conclusions: This new technology combines the reproducibility of competitive PCR with accurate quantitative detection by in-tube melting-curve analysis, enabling efficient analysis of mRNA profiles in samples with small numbers of cells or small amounts of tissue, as well as in archival FFPE tissues.






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