Quantitative Reverse Transcription-PCR Assay with an Internal Standard for the Detection of Prostate-specific Antigen mRNA

Abstract

Background: Circulating prostate cells can be detected with a reverse transcription-PCR (RT-PCR) assay for prostate-specific antigen (PSA) mRNA. We have developed a new quantitative RT-PCR method for measuring PSA mRNA. Methods: The method uses a PSA-like internal standard (IS) mRNA that is added into the sample at the beginning of the RNA extraction and coamplified by RT-PCR with the PSA in the sample. After PCR amplification, the IS and PSA products are selectively detected by hybridization in a microtitration plate using probes labeled with fluorescent europium chelates. Results: The method was validated with PSA and IS mRNAs and PSA-expressing cells to obtain a detection limit of 50 PSA mRNA copies (i.e., signal 2 times the mean of zero signal), linearity up to 106 copies, and detection of a single PSA-expressing cell. In preliminary evaluations, 60% (n = 10) of the prostate cancer patients with skeletal metastases gave results above the detection limit (500 PSA mRNA copies in 5 mL of blood). The total number of PSA copies ranged from 900 ± 200 to 44 100 ± 4900 (mean ± SD) in the samples, corresponding to ∼1–100 PSA-expressing cells in 5 mL of blood. In the controls (n = 34), none of the healthy females and 2 of 19 healthy males had detectable PSA mRNA [700 ± 100 and 2000 ± 900 (mean ± SD) PSA mRNA copies in 5 mL of blood for the 2 males]. Conclusions: The assay provides sensitive and quantitative detection of PSA mRNA expression from blood samples and can be used to establish the clinically significant number of PSA mRNA copies in prostate cancer.

Dual-Label Detection of Amplified Products in Quantitative RT-PCR Assay Using Lanthanide-Labeled Probes

“…The synthesis of PCR primers, detection probes, and DNA targets, along with the biotinylation of the 3 ′ PCR primer and the DNA targets, was carried out as reported previously (Table 1) (44). The detection probes for the hybridization assay contained additional diaminohexanedeoxycytidines (modC) to be labeled with lanthanide chelate as described earlier (5,12,36).…”

“…A constant amount of IS mRNA (5 ×10 4 molecules) was added into each sample after denaturation of the cells. The full-length PSA-like IS mRNA was produced in vitro and purified as described earlier (44). After TRI ZOLand 1-Bromo-3-Chloropropane (Sigma, St. Louis, MO, USA) treatment, RNA in the aqueous phase was precipitated once with isopropanol.…”

“…The long decay time allows the detection of the specific fluorescence after a time delay, during which the rapidly decreasing background fluorescence from the sample dies out. We have previously utilized TRF with single-label detection of the products amplified in quantitative RT-PCR (QRT-PCR) for prostate-specific antigen (PSA) mRNA (44). The use of different lanthanide chelates permits the detection of multiple analytes in the same reaction because of the sharp emission peaks and the different decay times of the lanthanides (Figure 1).…”

Blood and serum substances for markers of prostate cancer

“…The RT-PCR result is shown to be influenced by manipulation of the prostate such as biopsy, radical prostatectomy, and transurethral resection. In the future, quantitative RT-PCR with an internal standard may help to distinguish the low number of RNA messages from non-prostatic sources (Ylikoski et al, 1999).…”

Gene expression in HCV-associated HCC-identification of an upregulated gene encoding a protein related to ubiquitin conjugating enzyme

“…For quantification of one sample, multiple tubes are required and the amplification signal of the target is compared with that obtained from various known amounts of IS(Lundeberg et a l, 1991). For more accurate quantification of mRNA copies, an IS mRNA is added into the studied sample at the beginning of the RNA extraction step to avoid variations in the reverse transcription step(Ylikoski et al, 1999).…”