Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I

Real-time quantitative reverse transcriptase-polymerase chain reaction (RT- PCR) is the method of choice for rapid and reproducible measurements of cyt okine or growth factor expression in small samples. Fluorescence detection methods for monitoring real-time PCR include fluorogenic probes labelled wi th reporter and quencher dyes, such as Taqman probes or Molecular Beacons a nd the dsDNA-binding dye SYBR Green I. Fluorogenic (Taqman) probes for a ra nge of human and rat cytokines and growth factors were tested for sensitivi ty and compared with an assay for SYBR Green I quantification using real-li me fluorescence monitoring (PE Applied Biosystems Model 7700 sequence detec tor). SYBR Green I detection involved analysis of the melting temperature o f the PCR product and measurement of fluorescence at the optimum temperatur e. Fluorogenic probes provided sensitive and reproducible detection of targ ets that ranged from low (<10 copies/reaction) to high (>10(7) copies/react ion) expression. SYBR Green I gave reproducible quantification when the tar get gene was expressed at moderate to high levels (greater than or equal to 1000 copies/reaction), but did not give consistently reproducible quantifi cation when the target gene was expressed at low levels. Although optimizat ion of melting temperature improved the specificity of SYBR Green I detecti on, in our hands it did not equal the reproducible sensitivity and specific ity of fluorogenic probes. The latter method is the first choice for measur ement of low-level gene expression, although SYBR Green I is a simple and r eproducible means to quantify genes that are expressed at moderate to high levels.