Real-competitive PCR (rcPCR) has been shown to have high sensitivity, reproducibility and highthroughput potential. We describe significant development and evaluation of this methodology as a tool for measuring relative and absolute nucleic acid abundance within a cell at high throughput and low cost. Modifications to the original protocol allow analysis of gene expression levels using standard conditions regardless of mRNA abundance and assay type, thus increasing throughput and ease of reaction set-up while decreasing optimisation time. Additionally, we have developed a software package (TITAN) to automatically analyse the results. The details are relevant to researchers performing competitive PCR using any detection technique. The effectiveness of the described developments is demonstrated using 12 genes known to have differential expression in cell lines grown under normal and hypoxic conditions. Quantitative and qualitative comparisons to real-time PCR are presented. We also demonstrate that the technique is capable of detecting submicroscopic chromosomal DNA deletions
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