Absolute quantification of human chorionic gonadotropin-beta mRNA with TaqMan detection. 4.
ABSTRACT 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.
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ABSTRACT: To confirm the ectopic production of human chorionic gonadotropin (hCG) in lung cancer, we attempted to detect the presence of mRNA transcripts of the α and β genes for hCG in lung cancer tissues obtained from surgical operations. Although we were able to show the presence of hCGβ mRNA transcripts in lung cancer tissue by Northern blot, the sensitivity of the assay was too low for a precise analysis of hCGB mRNA transcripts in most lung cancers. Using reverse transcription PCR (RT-PCR) and Southern blot analysis, however, various amounts of mRNA transcripts of hCGβ genes 3, 5, 7 and 8 were demonstrated in 9 of the 14 lung cancer tissues examined, while no mRNA transcripts were detectable in 12 normal lung tissues from the same patients. Our results are consistent with a clear difference in serum and urinary hCGβ levels observed between normal subjects and lung cancer patients. The expression of the hCGα gene, however, was detected in normal lung tissues more frequently than in lung cancer tissues using RT-PCR Southern blot. Our results strongly suggests the production of hCGβ as being part of the phenotype of malignantly transformed lung cells and further strengthen its superior specificity over intact hCG or hCGα as a tumor marker for lung cancers. Int. J. Cancer 71:539-544, 1997. © 1997 Wiley-Liss, Inc.International Journal of Cancer 05/1997; 71(4):539 - 544. · 6.20 Impact Factor
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ABSTRACT: We describe a PCR-based assay for determining c-erbB-2 oncogene amplification in breast cancer in which we use the TaqMan system. Two fluorogenic probes anneal to the target between primers for c-erbB-2 and beta-globin genes and contain both a reporter dye (6-carboxy-fluorescein) and a quencher dye (6-carboxy-tetramethyl-rhodamine). During the extension phase of the PCR cycle, the 5'-->3' exonuclease activity of Taq polymerase cleaves the hybridized fluorogenic probe, resulting in an increase of fluorescence emission of the reporter dye that is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. Assay performance showed adequate precision and a lower detection limit and good correlation with the results obtained in the same samples by a competitive PCR assay (n = 25, r = 0.94, P < 0.01). This homogeneous assay is time-saving, avoids usually cumbersome postamplification procedures (that can be additional sources of inaccuracy and contamination), and seems suitable for determination of c-erbB-2 oncogene amplification in tumor specimens.Clinical Chemistry 06/1997; 43(5):752-8. · 7.15 Impact Factor
Chapter: Quantitative (real-time) PCR12/2004: pages 105-115;