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%-
Accurate analysis of mRNA expression levels of SNPs, highly homologous genes, and splicing variants requires techniques capable of quantifying low-copy-number mRNAs differing at single nucleotide positions. We have used an RT-PCR-based technique based on co-amplification of closely related target mRNA transcripts and assessed the effect of the stochastic distribution of low-copy-number templates on sampling variation when quantifying rare mRNA transcripts. The technique was optimized for maximal sensitivity to enable the analysis of samples containing a subpopulation of target cells and small microdissected samples. We demonstrate that the input level of template molecules is a critical determinant of the achievable assay precision. A minimum of approximately 50 molecules of template is required to discriminate between 2-fold differences in the expression levels of two transcripts. At levels above 1000 molecules of input template, the stochastic effects on sampling variation become negligible.
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