Targeted gene-expression analysis by genome-controlled reverse transcription-PCR.
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.
Stenman
Jakob
J
Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland. jakob.stenman@helsinki.fi
Paju
Annukka
A
Rissanen
Oso
O
Tenkanen
Tuomas
T
Haglund
Caj
C
Räsänen
Jari
J
Salo
Jarmo
J
Stenman
Ulf-Håkan
UH
Orpana
Arto
A
D001471:Barrett Esophagus / Q000175:diagnosis / Q000235:genetics; D015415:Biomarkers / Q000032:analysis; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003110:Colonic Neoplasms / Q000175:diagnosis / Q000235:genetics*; D015870:Gene Expression; D016678:Genome; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D015415:Biomarkers; D012333:RNA, Messenger