Development and Validation of an Externally Standardised Quantitative Insulin-like Growth Factor-1 RT-PCR Using LightCycler

RT-PCR has a detection limit 10–100 fold lower than protection-assay or northern hybridisation, respectively. The RT-ribonuclease PCR quantification technique of choice depends on the target sequence, the expected range of the mRNA amount present in the tissue, the degree of accuracy required, and whether quantification needs to be relative or absolute. Externally standardised RT-PCR with quantification on ethidium bromide-stained gels followed by densitometry is widely used, but the degree of accuracy is limited, and the quantification is more relative than absolute. For an exact quantitative measurement of low abundant gene expression, only a few PCR methods allow reliable mRNA quantification. At present, the following RT-PCR methods are suitable for sensitive quantification: