Received on May 4, 2006
Accepted on August 30, 2006

Molecular Diagnostics and Genetics

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

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

^* To whom correspondence should be addressed. 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.