Improved Biplex Quantitative Real-Time Polymerase Chain Reaction with Modified Primers for Gene Expression Analysis

Stabilizing modified bases incorporated in primers allows the reduction of housekeeping gene primer concentration not possible with regular primers without sacrificing amplification efficiency. Low primer concentration allows coamplification of the most abundant housekeeping genes with very rare templates without mutual inhibition. Real-time polymerase chain reaction (PCR) coamplification of 18S ribosomal RNA with several genes of interest was used in this study with MGB Eclipse® (Nanogen, San Diego, CA) hybridization probes. The results may be useful for high throughput gene expression studies as they simplify validation experiments.