Quantitative analysis of gene expression using PCR

A method of quantitation of expression of a first and a second target protein-encoding nucleic acids using a PCR based assay in which amplification of nucleic acid is detected by release of a reporter fluorescent signal which is measured as a normalised reporter value (“ΔRn”), the method comprising:

• providing a limiting concentration of a first primer pair, the first primer pair being adapted for amplification of a desired first target, the limiting concentration being selected to provide a first ΔRn value as measured by release of a first fluorescent reporter compound from a first reporter probe;
• selecting a non-limiting concentration of a second primer pair and probe set, the second primer pair being adapted for amplification of a desired second target, the amplification being measured by release of a second fluorescent reporter compound from a second reporter probe, the concentration being selected to provide a second ΔRn value of from three to forty times that provided by said first reporter compound;
• bringing together said first and second primer pairs and probes at their limited and selected concentrations respectively into contact with a sample of nucleic acid;
• performing a polymerase chain reaction under conditions in which the release of the first and second reporter compounds is measured;
• and
• quantitating the amount of first and second target nucleic acids.