Analytical variables of reverse transcription-polymerase chain reaction-based detection of disseminated prostate cancer cells

Early systemic spread of occult tumor cells that may develop into founders of incurable distant metastasis has been identified in prostate cancer pati ents by reverse transcription-PCR (RT-PCR) amplification of prostate-specif ic antigen (PSA) mRNA, Nevertheless, the introduction of this new staging t ool into the clinical setting has been hampered by the disparate and contra dictory data on the sensitivity and specificity of RT-PCR methods reported recently, We used PSA RT-PCR to examine the influence of analytical variabl es such as priming and enzyme of reverse transcriptase reaction, temperatur e and time of primer annealing, primer extension and denaturation, as well as the concentrations of magnesium chloride, Tag polymerase, deoxynucleotid e triphosphate, primers and BSA on the amplification process, By systematic ally varying these chemical and physical components, we could demonstrate a significant increase in amplification yield and in stringency of primer an nealing, This may explain the wide variety of published findings on molecul ar staging of prostate cancer, which currently impedes the clinical introdu ction of PSA RT-PCR assays in prostate cancer. Methodological analyses are needed for standardization and quality assurance to achieve reproducible mo lecular methods that can be used in clinical practice.