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Clinical Chemistry 44: 2441-2445, 1998;
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(Clinical Chemistry. 1998;44:2441-2445.)
© 1998 American Association for Clinical Chemistry, Inc.

Enzymes and Protein Markers

Quantitative reverse transcription-PCR assay of the RNA component of human telomerase using the TaqMan fluorogenic detection system

Tomomi Yajima1, Atsuhito Yagihashi1, Hidekazu Kameshima1, Daisuke Kobayashi1, Daisuke Furuya1, Koichi Hirata2 and Naoki Watanabe1,a

Departments of
1 Laboratory Diagnosis and
2 Surgery, Sapporo Medical University School of Medicine, Sapporo 060-8543, Japan.
a Address correspondence to this author at: Department of Laboratory Diagnosis, Sapporo Medical University School of Medicine, South-1, West-16, Chuo-ku, Sapporo 060-8543, Japan. Fax 81-11-622-7502; e-mail watanabn{at}sapmed.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We established the validity of a quantitative reverse transcription (RT)-PCR assay for the RNA component of human telomerase (hTR), using the TaqMan fluorogenic detection system. Using this assay, we quantified hTR expression in two human pancreatic cancer cell lines, ASPC-1 and MIAPaCa-2. Our results indicated that hTR expression in MIAPaCa-2 was 1.99-fold higher than that in ASPC-1 cells. This TaqMan RT-PCR assay appears to be useful in determining the amount of hTR in clinical specimens.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Telomerase is an enzyme that synthesizes and adds repetitive telomeric sequences of (TTAGGG)n to ends of chromosomes. Telomerase activity has been found in 85% of human cancers, including stomach, breast, bladder, colon, prostate, and liver, and in immortalized cell lines but not in most nondiseased tissues (1)(2)(3)(4)(5)(6)(7). Therefore, telomerase is considered closely related to attainment of cellular immortality (one of the steps in carcinogenesis) as well as cellular senescence. Because telomerase-associated genes such as those encoding the human telomerase RNA component (hTR),1 human telomerase reverse transcriptase, and telomerase-associated protein-1 have been cloned, it has become possible to study the expression of these genes (8)(9)(10)(11)(12).

Comparison of telomerase activities and expression of the associated genes should help clarify the regulation of telomerase activity. However, hTR expression is detected in cancer as well as in nondiseased tissues, and development of quantitative assays for hTR is required (8)(13)(14)(15). In addition, telomerase activity and expression of the associated genes have been defined only as positive or negative and not quantified to relate telomerase activity and gene expression. Recently, TaqMan fluorescent chemical analysis has been applied to PCR (16)(17)(18). This method is based on use of the 5' nuclease activity of Taq polymerase to cleave a nonextendable dual-labeled fluorogenic hybridization probe during the extension phase of PCR. TaqMan analysis makes quantitative PCR or reverse transcription (RT)-PCR (19, 20) attainable. Thus, we established a quantitative assay for hTR expression in two human pancreatic cancer cell lines, ASPC-1 and MIAPaCa-2, using TaqMan RT-PCR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
cells
Human pancreatic cancer cell lines were obtained from the American Type Culture Collection. ASPC-1 cells were cultured in RPMI-1640 (Nipro) supplemented with 200 mL/L heat-inactivated fetal calf serum (Biological Industries), and MIAPaCa-2 cells were cultured in Dulbecco's modified Eagle's medium (Nipro) supplemented with 100 mL/L heat-inactivated fetal calf serum.

total rna extraction
Cells were trypsinized, and the cell pellets were collected by centrifugation at 1000g for 5 min at 4 °C. SepaGene RV-R (Sanko Pure Chemicals) was used to extract total RNA from cells; this extract was assayed for RNA with Gene Quant DNA/RNA (Pharmacia).

primers and probes
Primers and the TaqMan probe for hTR were designed using the primer design software Primer ExpressTM (Perkin-Elmer Applied Biosystems). Synthesis of this probe and primers was performed by Perkin-Elmer Japan and by Tanaka. Primers and the TaqMan probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; TaqMan GAPDH control reagent kit) also were purchased from Perkin-Elmer Applied Biosystems. Table 1 shows the sequences for the TaqMan probes and primers used.


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  		Table 1. Primers and probes.

AmpliTaq DNA polymerase extended the primer and displaced the TaqMan probe through its 5'-3' exonuclease activity. The probes were labeled with a reporter fluorescent dye [6-carboxy-fluorescein (FAM) or 2,7-dimethoxy-4,5-dichloro-6-carboxy-fluorescein (JOE)] at the 5' end and a quencher fluorescent dye (6-carboxy-tetramethyl-rhodamine) at the 3' end. Nuclease degradation of the hybridization probe removed the quenching effect of 6-carboxy-tetramethyl-rhodamine from the FAM or JOE fluorescent emission, increasing the peak fluorescent emission at 517 and 554 nm, respectively. No signal was emitted when the probe was intact.

one-step rt-pcr
Fifty microliters of reaction mixture were used, containing 10 ng of the extracted total RNA, 1x TaqMan buffer A, 5.5 mmol/L MgCl2, 300 µmol/L dATP, dGTP, and dCTP, 600 µmol/L dUTP, 0.2 µmol/L forward and reverse primers, 0.1 µmol/L TaqMan probe, 1.25 U of AmpliTaq Gold, 12.5 U of MuLV reverse transcriptase, and 20 U of RNase Inhibitor (Perkin-Elmer Applied Biosystems). The conditions of one-step RT-PCR were as follows: 30 min at 48 °C (stage 1, RT), 10 min at 95 °C (stage 2, RT inactivation and AmpliTaq Gold activation), and then 40 cycles of amplification for 15 s at 95 °C and 1 min at 60 °C (stage 3, PCR). The assay used an instrument capable of measuring fluorescence in real time (ABI Prism 7700 Sequence Detector; Perkin-Elmer Applied Biosystems). Signals were detected according to the manufacturer's instructions. A computer algorithm compared the amount of reporter dye emission (R) with the quenching dye emission (Q) during the PCR amplification, generating a {Delta}Rn value as follows: {Delta}Rn = (Rn+) - (Rn-), where Rn+ is (emission intensity of reporter)/(emission intensity of quencher at any given time in the reaction tube), and Rn- is (emission intensity of reporter)/(emission intensity of quencher measured before PCR amplification in the same reaction tube). The {Delta}Rn value is the emission of the reporter over the starting background fluorescence and is used for construction of amplification plots.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
amplification plots and cycle threshold
hTR and GAPDH amplification plots for four different dilutions (containing 100, 50, 10, or 5 ng) of total RNA from ASPC-1 and samples of unknown total RNA concentration from ASPC-1 and MIAPaCa-2 are shown in Fig. 1 . ASPC-1 samples were used as the basis for comparing results and calibration curves. Amplification plots were used to determine the threshold cycle (CT). To determine the CT values and obtain the linearity of the calibration curves, the optimal {Delta}Rn was selected in the exponential phase of amplification plots for each dilution of total RNA. The CT value represented the PCR cycle at which an increase in reporter fluorescence above the line of the optimal {Delta}Rn was first detected. Experientially, this line was drawn at a {Delta}Rn of 0.05 (usually 10 SD of the baseline) (20).



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  		Figure 1. Amplification plots of hTR (top) and GAPDH (bottom).

Four different dilutions, including 100 ng (red), 50 ng (blue), 10 ng (green), or 5 ng (pink) of total RNA from ASPC-1, and unknown sample concentrations (yellow and purple) were tested in triplicate. The unknown samples were obtained from the total RNA extract of MIAPaCa-2 and ASPC-1 cells. Amplification plots were used to determine the CT; the xy plots represent the PCR cycle number as x and the mean {Delta}Rn value as y. To determine the optimal CT, the optimal {Delta}Rn (y = 0.05) was selected in the exponential phase of amplification plots for each dilution of total RNA (See Results for details).

calibration curves
For relative quantification of hTR expression, a calibration curve was constructed. RNA for GAPDH, a "housekeeping" enzyme, was used as an endogenous control. Fig. 2 shows calibration curves for hTR and GAPDH RNA quantitation in ASPC-1 cells as detected using FAM- and JOE-labeled probes. The calibration curve as an xy (scatter) plot represented the log of the input amount (log ng of total starting RNA) as x and CT as y. Equations were derived from the lines of the calibration curves. The two formulas for log ng of hTR and GAPDH were as follows: hTR, y = -3.26x + 28.63 (r = 0.999); GAPDH, y = -3.19x + 26.46 (r = 0.997). When the CT value of a sample was substituted into the formula for hTR or GAPDH, the concentration of hTR or GAPDH could be calculated.



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  		Figure 2. Calibration curves for hTR (top) and GAPDH (bottom).

RNA quantification of ASPC-1 was detected using FAM- and JOE-labeled probes. The calibration curve of the xy (scatter) plot represents log ng of total starting RNA as x and CT as y. Equations were derived from the lines of the calibration curves. The two formulas for log ng of hTR and GAPDH are as follows: hTR, y = -3.26x + 28.63 (r = 0.999); GAPDH, y = -3.19x + 26.46 (r = 0.997). When the CT value of a sample is substituted as y into the formula for hTR or GAPDH, the concentration of hTR or GAPDH can be calculated.

data analysis
Table 2 shows the mean concentrations of hTR and GAPDH found in ASPC-1 and MIAPaCa-2 in triplicate determinations of each sample, together with normalized concentrations of hTR. To normalize for differences in the amount of total RNA added to each reaction, GAPDH was selected as an endogenous RNA control. The normalized concentration of hTR, an arbitrary number that can be used to compare the relative amounts of hTR in different samples, was determined by dividing the concentration of hTR by the concentration of GAPDH. Our results indicated that hTR expression in MIAPaCa-2 was 1.99-fold higher than in ASPC-1 cells.


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  		Table 2. Detection of hTR and GAPDH in ASPC-1 and MIAPaCa-2 cell lines.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We developed a quantitative RT-PCR assay of hTR, using a TaqMan fluorogenic detection system. In previous reports, hTR was examined using RT-PCR or Northern blots. (8)(9)(13)(14)(15) To make the RT-PCR more quantitative using the conventional internal standard method, it was necessary to determine the optimal cycle number and sample concentration to compare the amounts of amplification products in the exponential amplification phase of PCR. This implies that the PCR assay should be performed in serial cycles on serially diluted samples. A large amount of mRNA and a lengthy series of steps (mRNA preparation, electrophoresis, blotting, hybridization, and autoradiography) are needed to perform Northern analysis, requiring >1 day. In the present assay using the TaqMan fluorogenic detection system, the amplification signals are detected in real time, permitting accurate quantification of amounts of initial RNA template accurately because the system can select signals easily in the exponential amplification phase of PCR. In addition, hTR can be analyzed in a single tube, from RT-PCR to detection, in ~3 h.

Because the TaqMan probe specifically hybridizes with its target, a sequence-specific amplification signal was detected. Our data demonstrated that hTR expression in MIAPaCa-2 cells was 1.99-fold higher than in ASPC-1. Additional studies are needed to determine whether this difference is clinically significant. In addition, we have found in preliminary studies that hTR expression is measurable using TaqMan RT-PCR in clinical gastric mucosal specimens, including those showing gastric cancer, intestinal metaplasia, atrophic gastritis, and nondiseased gastric mucosa (unpublished data). hTR expression was detected in cancer as well as in nondiseased tissues. Therefore, to clarify the relationship between hTR expression and telomerase activity, a quantitative assay for hTR is required. In addition, known quantities of hTR can be used to construct the calibration curve, and thus normalization is not necessary.

In conclusion, the TaqMan RT-PCR assay is a rapid, accurate method for measuring hTR expression and shows promise for the quantification of hTR in clinical specimens.


   Acknowledgments
 
We thank Osamu Sakatsume of Perkin-Elmer Applied Biosystems, Japan for probe synthesis and informative discussions concerning the use of the ABI Prism 7700 detection system.


   Footnotes
 
1 Nonstandard abbreviations: hTR, human telomerase RNA component; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FAM, 6-carboxy-fluorescein; JOE, 2,7-dimethoxy-4,5-dichloro-6-carboxy-fluorescein; and CT, threshold cycle.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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