Double-differential PCR for gene dosage estimation of erbB oncogenes in benign and cancer tissues and comparison to cellular DNA content

摘要: Competitive and differential quantitative PCR methods circumvent the limiting factors of which cause poor reproducibility. We describe development performance evaluation another method, double-differential (ddPCR). The ddPCR method comprises co-amplification single-copy gene HBB, erbB-1, erbB-2 erbB-3 oncogenes second reference SOD2 under equal reaction conditions. ratio band intensities products in silver-stained polyacrylamide gels expresses average copy number (AGCN) per cell erbB oncogenes. coefficient variability (CV) was less than 25% for an AGCN 1. data were correlation to results from dot blotting. DNA image analysis did not reveal any between content dosage deviation applied normal breast tissue, benign diseases, cancer tissue lymph node metastases. suggest this as being reproducible, low cost rapid, therefore suitable clinical studies on oncogene estimation.