High-resolution semi-quantitative real-time PCR without the use of a standard curve

The repeatability and sensitivity of a simple, adaptable, semi-quantitative , realtime RT-PCR assay was investigated. The assay can be easily and rapid ly applied to quantitate relative levels of any gene product without using standards, provided that amplification conditions are specific for the PCR product of interest. Using the Light-Cycler (TM) real-time PCR machine, a s erial 10-fold dilution series, (spanning four orders of magnitude) of a 379 -bp cDNA template was amplified, and the PCR product was defected using SYB R (R) Green I chemistry. The experiment was repeated on a subsequent day. T he experimental design was such that the data lent itself to analysis using an appropriate method for testing repeatability. It was found that, within a single assay for samples assayed in triplicate, a difference of 23% may be reliably detected Furthermore, when all of the factors that contribute t o variability in the assay are taken into account, such as day-to-day varia tion in pipetting and amplification efficiency, a 52% difference in target template can be detected using a sample size of 4. The assay was found to b e linear over at least four orders of magnitude.