Purpose: During the last decade numerous different reverse transcriptase-polymerase chain reaction (RT-PCR) techniques have been described. However, the lack of highly sensitive, quantitative and reliable methodology has been responsible for its limited use in modern urology. Early semiquantitative RT-PCR techniques often proved not to produce consistent results and have a high failure rate due to complicated working models. In this article we provide a comprehensive and intelligible description of real-time PCR technology, which is a novel quantitative methodology to analyze gene expression. In addition, we report the first preclinical and clinical applications in molecular urology.
Materials and methods: The current literature was reviewed in regard to different current real-time RT-PCR protocols and their use in modern urological oncology.
Results: Real-time RT-PCR is a reliable, rapid and relatively inexpensive technique that can be easily adapted for standardized preclinical and clinical applications at different centers. Its sensitivity equals at least that of conventional RT-PCR and the option of exact quantification of gene expressions allows proper differentiation among high, low and illegitimate RNA transcription. It eliminates post-PCR processing of PCR products, thereby, increasing throughput and decreasing the chance of carryover contamination.
Conclusions: Although the application of real-time RT-PCR has gained wide acceptance in urological research, its routine clinical use is still in its infancy. However, due to its high sensitivity and exact quantitation real-time RT-PCR may be the method of choice for modern preclinical and clinical studies in the future.