Absolute quantification of human chorionic gonadotropin-beta mRNA with TaqMan detection. 4

We describe a reverse transcriptase-polymerase chain reaction (RT-PCR) for determination of human chorionic gonadotropin-beta (HCG beta) mRNA copies using the TaqMan system. To evaluate our quantitative assay, we analyzed HCG beta transcripts of all protein coding genes (HCG beta 5, 3, 8, and 7) in human RNA panels of different normal tissues and in glycodelin-A-stimulated trophoblast cell cultures. Absolute quantification using HCG beta TaqMan probe was found to be highly reproducible. Our study of RNA panels confirms recently published results that expression of HCG beta transcripts is a common feature of a great variety of different normal tissues. High levels of HCG beta mRNAs (> 1,000 molecules per 200 ng RNA) were detected in placenta, uterus, and testis. An increase of HCG beta mRNA expression (1.7-fold) was detected at 150 micrograms/mL glycodelin-A treatment in trophoblast cell culture. Time-dependence study showed that the increase in HCG beta mRNA level was evident at 60 min after glycodelin-A treatment. In summary, we have developed a highly sensitive one-tube, one-enzyme quantitative RT-PCR system that is time-saving and avoids postamplification procedures.