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Clinical Cancer Research Vol. 9, 3338-3344, August 2003
© 2003 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Development of a Real-Time Polymerase Chain Reaction Assay for Prediction of the Uptake of Meta-[131I]iodobenzylguanidine by Neuroblastoma Tumors1

Sean Carlin2, Rob J. Mairs3, Anthony G. McCluskey, Deborah A. Tweddle, Alan Sprigg, Christine Estlin, Julian Board, Rani E. George, Caroline Ellershaw, Andrew D. J. Pearson, John Lunec, Paolo G. Montaldo, Mirco Ponzoni, Berthe L. van Eck-Smit, Cees A. Hoefnagel, Marieke D. van den Brug, Godelieve A. M. Tytgat and Huib N. Caron

Departments of Radiation Oncology and Child Health, University of Glasgow, Cancer Research UK Beatson Laboratories, Glasgow G61 1BD, United Kingdom [S. C., R. J. M., A. G. M.]; Department of Child Health, Royal Victoria Infirmary and University of Newcastle, Newcastle upon Tyne NE1 4LP, United Kingdom [D. A. T., A. D. J. P.]; Cancer Research Unit, The Medical School, University of Newcastle, Newcastle NE2 4HH, United Kingdom [D. A. T., R. E. G., A. D. J. P., J. L.]; Department of Radiology, Sheffield Children’s Hospital NHS Trust, Sheffield S10 2TH, United Kingdom [A. S., C. Es., J. B., R. E. G.]; United Kingdom Children’s Cancer Study Group, Department of Epidemiology and Public Health, University of Leicester, Leicester LE1 6TP, United Kingdom [C. El.]; Pediatric Oncology Research Laboratory, G. Gaslini Institute, 16148 Genoa, Italy [P. G. M., M. P.]; Department of Nuclear Medicine, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital, Amsterdam, the Netherlands [C. A. H.]; and Departments of Nuclear Medicine [B. L. v. E-S.] and Pediatric Oncology [M. v. d. B., G. A. M. T., H. N. C.], Academical Medical Center, University of Amsterdam, 1100 DD Amsterdam, the Netherlands


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: The suitability of neuroblastoma patients for therapy using radiolabeled meta-iodobenzylguanidine (MIBG) is determined by scintigraphy after the administration of a tracer dose of radioiodinated MIBG whose uptake is dependent upon the cellular expression of the noradrenaline transporter (NAT). As a possible alternative to gamma camera imaging, we developed a novel molecular assay of NAT expression. mRNA extracted from neuroblastoma biopsy samples, obtained retrospectively, was reverse transcribed, and NAT-specific cDNA was quantified by real-time PCR, referenced against the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase.

Experimental Design: Tumor specimens from 54 neuroblastoma patients were analyzed using real-time PCR, and NAT expression was compared with the corresponding diagnostic scintigrams.

Results: Forty-eight of 54 (89%) of tumors showed MIBG uptake by scintigraphy. NAT expression was found to be significantly associated with MIBG uptake (P < 0.0001, Fisher’s exact test). None of the samples from the six tumors that failed to concentrate MIBG expressed detectable levels of the NAT (specificity = 1.0). However, of the 48 MIBG uptake-positive tumors, only 43 (90%) expressed NAT (sensitivity = 0.9). The real-time PCR test has a positive predictive value of 1.0 but a negative predictive value of 0.55.

Conclusions: The results indicate that whereas this method has substantial ability to predict the capacity of neuroblastoma tumors to accumulate MIBG, confirmation is required in prospective studies to determine more accurately the predictive strength of the test and its role in the management of patients with neuroblastoma.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A total of 85–90% of neuroblastoma tumor cells express the NAT,4 a 12-spanning integral membrane protein responsible for the active intracellular accumulation of catecholamine neurotransmitters. [131I]MIBG, a structural analogue of the adrenergic neurone blockers guanethidine and bretyllium (1) that is also accumulated by NAT-expressing cells (2) , is a useful agent for the diagnosis and treatment of neuroblastoma (3) . Previous studies have demonstrated that NAT expression correlated with [131I]MIBG uptake in vitro (4) .

Clinical diagnosis of neuroblastoma involves histological examination of biopsy specimens and evaluation of scintigrams obtained after the administration of a tracer dose of either [123I]MIBG or [131I]MIBG. Tumor uptake of MIBG at this stage also indicates suitability of the patient for a treatment regimen that includes [131I]MIBG therapy. However, radioiodinated MIBG is not available immediately to most hospitals, so that the time of admission to gamma camera scanning and reporting may take more than 1 week. A rapid and sensitive molecular assay for MIBG active uptake, which could be applied at initial biopsy, would be invaluable in selecting patients for therapeutic strategies that incorporate radiolabelled MIBG.

Whereas quantitative PCR is feasible using serial dilution of cDNA followed by analysis of PCR end points, this procedure has proven both time-consuming and poorly reproducible (5) . The recent development of fluorescent TaqMan methodology shows great promise and may supersede existing methods. The TaqMan method of quantitative PCR utilizes the 5'-exonuclease activity of Taq polymerase to cleave a specific fluorogenic oligonucleotide probe during the extension phase of the reaction (6) . Fluorescence emission is measured in real-time after every PCR cycle. The CT value of product detection, used for quantitation, is derived from the exponential phase of the PCR reaction, rather than the plateau phase, where increasing cycle number has no effect on product formation. This results in greater accuracy and sensitivity over alternative methods of nucleic acid quantitation. These features should facilitate progress toward the clinical application of PCR-based assays. Moreover, a RT-PCR analysis of tumor expression of NAT-specific transcripts can be completed in one half of a working day. Therefore, this procedure has the potential to allow the prompt identification of patients who may benefit from targeted therapy.

Using real-time PCR, our goals were (a) to establish the relationship between the level of NAT gene transcription by cell lines and their capacity for active uptake of MIBG and (b) to determine whether the detection of NAT cDNA sequences in neuroblastoma biopsy samples could predict the uptake by tumors of MIBG.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Culture Conditions.
Five human cell lines of neurogenic origin were cultured: SK-N-SH; SK-N-BE(2C); SH-SY5Y; SK-N-MC (7) ; and IMR-32 (8) . In addition, two non-neural crest-derived control cell lines were evaluated. These were the human breast adenocarcinoma line MCF-7 (9) and the human glioma cell line UVW, which was established in this laboratory (10) . Cells were maintained in either MEM (SK-N-SH, IMR 32, SH-SY5Y, SK-N-MC, and UVW) or RPMI 1640 [SK-N-BE(2C) and MCF-7], containing 10% FCS and glutamine (2 mM) at 37°C in a 5% CO2 atmosphere. All media and supplements were purchased from Gibco (Paisley, United Kingdom).

Tumor Samples.
Samples of 54 neuroblastoma tumors, frozen in liquid nitrogen immediately after excision, were obtained retrospectively from the Academical Medical Center (Amsterdam, the Netherlands), the G. Gaslini Institute (Genoa, Italy), and 10 United Kingdom Children’s Cancer Study Group hospitals. Histological examination revealed that all samples contained greater than 20% neuroblastoma cellularity. Biopsy material was obtained from the primary tumor in each case. Total RNA was extracted from cell lines and patient biopsy material using the RNazol method according to the manufacturer’s instructions (Biogenesis, Poole, United Kingdom). The RNA samples were assessed for NAT and GAPDH expression by reverse transcription and real-time PCR. This study was approved by the Research Ethics Committees of the participating institutions.

MIBG Uptake in Cell Lines.
No-carrier-added [131I]MIBG was synthesized by iododesilylation of meta-trimethylsilylbenzylguanidine according to the method of Vaidyanathan and Zalutsky (11) . The ability of cell lines to take up MIBG was assessed as described previously (12) . Briefly, cellular monolayers were incubated with 7 kBq of no-carrier-added [131I]MIBG, so that the drug concentration was less than 100 pM. After incubation for 2 h at 37°C, the cells were washed twice with ice-cold PBS, and radioactivity was extracted with two 0.5-ml aliquots of 10% (w/v) trichloroacetic acid. The activity of the combined extracts was measured using a gamma counter (Canberra Packard, Berkshire, United Kingdom).

Nonspecific uptake of [131I]MIBG was assessed in the presence of desmethylimipramine, a tricyclic antidepressant that inhibits the re-uptake of neurotransmitters by adrenergic neurons. To determine the percentage contribution to cellular accumulation of the radiopharmaceutical by the active uptake process, cells were preincubated for 30 min with 1.5 µM desmethylimipramine in the absence of MIBG. The medium was then replaced with one containing both drugs, and after 2 h, the uptake of radioactivity was measured as described above.

Evaluation of Scintigrams.
MIBG scintigrams were obtained from the referral centers. Hard copy images were appraised independently by two assessors. The activity pattern was compared with the known site of the primary tumor (as biopsied) and correlated with other cross-sectional imaging as necessary (e.g., where liver uptake was superimposed on a primary upper abdominal tumor mass). Review of the MIBG scintigrams and cross-sectional imaging was performed blind, without prior knowledge of the abundance of NAT-specific mRNA. Only samples from patients with evaluable MIBG scans were included in the study. The uptake of [123I]MIBG into the primary mass was recorded as negative or positive.

Primers and Probes.
Primer and probe sequences were designed from the published sequence for the hNAT (database source GenBank, accession number M65105) using the ABI prism PrimerExpress v1.0 software. Both primers and probe were custom synthesized (MWG-Biotech, Milton Keynes, United Kingdom). The sense primer (5'-CGCTTCCCCTACCTCTGCTA-3') corresponded to bases 241–260 of the hNAT sequence. The antisense primer (5'-AGATTTTCCAAACGGTGGCA-3') was complementary to bases 372–391 of the hNAT sequence. These primers generated a PCR product of 151 bp.

The internal probe (5'-CGGTGCCTTCTTGATCCCGTACACACT-3') corresponded to bases 273–299 of the hNAT sequence. The probe was labeled with the fluorescent reporter dye 6-carboxyfluoroscein at the 5' end and with the quencher molecule 6-carboxytetramethylrhodamine at the 3' end.

The housekeeping gene GAPDH was used as an internal standard for all real-time PCR reactions. Primers and probe were obtained from Perkin-Elmer Applied Biosystems (Cheshire, United Kingdom).

Standard Curve Construction.
NAT-specific and GAPDH-specific PCR products were obtained by reverse transcribing and PCR amplifying 1 µg of total RNA obtained from the human neuroblastoma cell line SK-N-BE(2C) using the same primer pairs used later in the quantitative real-time PCR. The resulting PCR products were then purified using an S-400 spin column (Pharmacia Biotech, Uppsala, Sweden) and quality assessed by agarose gel electrophoresis and ethidium bromide staining. The PCR product was quantified spectrophotometrically and then serially diluted in mouse genomic DNA (Clontech) to maintain a constant total DNA concentration. The standard curve used for quantitation of the real-time PCR reaction was constructed using 102 to 107 copies of the SK-N-BE(2C) PCR product obtained from the reaction described above.

Real-Time PCR Amplification.
One µg of total RNA was reverse transcribed in a 50-µl reaction volume, and 2.5 µl of the resulting solution, containing cDNA template, were added to an amplification reaction mixture (total volume, 25 µl) consisting of 5.5 mM MgCl2; 2.5 µl of 10x TaqMan buffer; 200 µM dATP, dCTP, and dGTP; 400 µM dUTP; 0.625 unit of AmpliTaq Gold; 0.25 unit of AmpErase uracil N-glycosylase; and 100 nM each of both primers and probe. The thermal cycling conditions consisted of an initial incubation for 2 min at 50°C, followed by 10 min at 95°C. Thermal cycling was then carried out at 95°C for 15 s followed by 60°C for 1 min for 40 cycles. Each assay included a standard curve (102 to 107 copies) and a no-template control, along with the unknown cDNA templates obtained from the reverse transcription step. PCR reactions were performed using an ABI prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems, Foster City, CA), which measured the fluorescent signal generated by the PCR reaction.

Statistical Analysis.
The expression of the NAT as a function of specific uptake of MIBG by neuroblastoma cells was determined by linear regression. The relationship between tumor uptake of MIBG and NAT gene expression was examined by Fisher’s exact test (two-sided) using Graph Pad Prism software, version 3.0 (1999).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Real-Time PCR.
A range of quantities of NAT- and GAPDH-specific PCR products were amplified using the real-time PCR method to establish corresponding CT values (Fig. 1A)Citation . These were plotted against the initial quantity of substrate to produce standard curves (Fig. 1B)Citation . This exemplifies the high sensitivity and accuracy of amplification over a large concentration range (r = 0.991). RNA obtained from cell lines and patient biopsy material was reverse transcribed and PCR amplified using the real-time methodology. Samples were assayed three times in duplicate for both NAT and GAPDH expression.



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  		Fig. 1. Amplification plot (a) and standard curve (b) of the NAT sequence. Standards were obtained by PCR amplification of cDNA from the cell line SK-N-BE(2C). The NAT-specific PCR product was then quantified spectrophotometrically, serially diluted, and amplified using the real-time PCR method. The plots, from left to right, correspond to 107 to 102 NAT sequence copies (a). The CT values obtained were plotted against the initial quantity of substrate to produce a standard curve (b). A similar procedure was used to quantify the reference GAPDH sequence.

 
[131I]MIBG Uptake Compared with NAT Gene Expression in Cell Lines.
Four of five neuroblastoma cell lines, but none of the non-neural crest-derived cell lines, demonstrated active uptake of MIBG. Real-time RT-PCR, with specificity for NAT and GAPDH sequences, indicated transcription of the NAT gene only in cell lines that actively accumulated MIBG (Table 1)Citation . The coefficients of variation of the CT values of the NAT-positive cell lines were 2.9% [SK-N-BE(2C)], 2.8% (SK-N-SH), 2.5% (SH-SY5Y), and 2.0% (IMR-32). These indices of reproducibility are superior to those obtained previously by means of conventional RT-PCR methodology (4) . The level of expression of the NATgene by this panel of five neuroblastoma cell lines correlated significantly (P < 0.001) with the capacity for active uptake of MIBG.


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  		Table 1 NAT gene expression and MIBG uptake by neuroblastoma cell lines

Using the RNA obtained from seven human tumor cell lines, NAT and GAPDH expression was assessed by reverse transcription and real-time PCR amplification. The results are means ± SD of three separate determinations in duplicate. Active uptake of [131I]MIBG was determined by subtraction of nonspecific uptake (assessed in the presence of desmethylimipramine) from total [131I]MIBG uptake.

 
Distribution of NAT Gene Expression in Biopsy Specimens.
The real-time PCR procedure revealed a wide range of NAT gene expression levels in neuroblastoma biopsy samples (Fig. 2)Citation . Most tumors (63%) expressed <2000 NAT copies/104 reference gene (GAPDH) transcripts. A minority of the tumors showed much higher NAT transcription levels. The highest expression of the cohort (two tumors) was >9000 NAT transcripts/104 GAPDH transcripts.



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  		Fig. 2. Distribution of NAT mRNA expression levels in neuroblastoma biopsy samples.

 
MIBG Uptake by Neuroblastoma Tumors Was Compared with NAT Gene Expression in Biopsies.
NAT and GAPDH copy numbers of neuroblastoma biopsy samples were obtained in the same manner as described for cell lines. RNA isolates from biopsy specimens were assayed three times in duplicate. Table 2Citation illustrates the disease stage and MIBG scintigraphic and RT-PCR results from the specimens collected from patients. Of the 54 patients’ samples with evaluable MIBG scans, 48 (89%) showed MIBG uptake into the primary tumor. Forty-three of 54 (80%) tumor samples were positive for NAT mRNA. Of the 11 of 54 (20%) tumor specimens in which NAT-specific sequences were undetectable, 6 of 54 (11%) were derived from tumors that did not accumulate MIBG, whereas 5 of 54 (9%) originated from MIBG scan-positive primary tumors. We did not observe NAT expression in samples from tumors that failed to accumulate MIBG (Table 3)Citation . There was a significant association between tumor MIBG uptake, evaluated by scintigraphy, and PCR-determined NAT expression (P < 0.0001, Fisher’s exact test). The prognostic value of a NAT-specific, positive PCR test was 1 (100%). However the predictive merit of a negative test was only 0.55 (55%).


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  		Table 2 Comparison of tumor uptake of MIBG with NAT mRNA expression in biopsy samples, determined by gamma camera scanning of the patients and RT-PCR, respectively

 

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  		Table 3 MIBG uptake by neuroblastoma tumors compared with NAT expression

The accumulation of MIBG by tumor was determined by gamma camera scanning after the administration to patients of diagnostic grade [123I]MIBG. NAT expression in neuroblastoma biopsy specimens was assessed by real-time PCR analysis.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have developed a rapid and specific assay for NAT expression in biopsy samples obtained from neuroblastoma patients. The assay, based on real-time PCR analysis, showed substantial power to predict MIBG uptake capacity and has the potential to select patients for MIBG therapy.

The procedure described in this report is superior, in terms of speed and reproducibility, to previously published methods of NAT mRNA quantitation (4) . The real-time PCR technique allows the simultaneous evaluation of a large number of individual samples as well as internal controls. This assay involves the use of industry-standardized reagents and requires less manipulation of sample than traditional PCR techniques, resulting in increased reliability and accuracy.

Of the 54 patients’ samples with evaluable MIBG scans, 48 (89%) showed MIBG uptake into the primary tumor, whereas 43 (80%) of the tumor samples were positive for the expression of NAT mRNA. This is equivalent to 90% concordance with respect to positive outcome of the two tests. All six tumors (11%) that failed to accumulate MIBG had undetectable NAT mRNA expression. However, biopsies from five patients (10.4%) with positive MIBG scans did not express detectable NAT mRNA. Therefore, whereas a positive PCR test always predicted tumor uptake of MIBG, a negative PCR test failed to correctly determine MIBG uptake capacity with a frequency of 45%. We conclude that PCR-based assessment of NAT gene expression cannot be regarded as fully predictive of MIBG uptake.

There are plausible explanations for the occasional coincidence of a negative PCR test with a positive MIBG scan. It is possible that the quality of biopsy material used for analysis was not optimal. In the present study, specimens were subjectively assessed for neuroblastoma content, and those with <20% neuroblastic component were rejected. It would be preferable to establish an objective assessment of the proportion of malignant cells in biopsy material. To predict tumor cell concentration of MIBG more accurately, it may be necessary to use alternative internal standards with specificity for neuroblastoma cells. Possibilities include the transcript of the tyrosine hydroxylase gene (13) , which is expressed only in catecholamine-synthesizing cells, or mRNA specific for neural crest cell proteins such as PGP 9.5 (14) or GD2 synthase (15) . Another possible reason is that monoamine transporters other than NAT may be partly responsible for the accumulation of MIBG by tumor cells. Although the principal mechanism of cellular uptake of MIBG involves the NAT, the possibility of tumor concentration via vesicular or cell membrane-associated monoamine transporters cannot be discounted (16 , 17) . Alternatively, mutant forms of NATcould be involved. These may be undetectable by the PCR primers used in this study.

A major obstacle to the application of molecular tests on real tumors rather than cultured cells is tumor heterogeneity with respect to expression of specific markers. To assess the sensitivity of the RT-PCR assay in vivo, it is necessary to compare MIBG concentration with real-time PCR data derived from tumors that contain known percentages of NAT-positive cells. Recently, we described an in vitro tumor model that incorporated cell population heterogeneity (18) . We prepared multicellular mosaic spheroids composed of variable percentages of two cell populations, one of which was transfected with the NAT gene. This model is now being adapted to address the limitation to detectability imposed by heterogeneity of NAT expression. To assess the sensitivity of the assay, we are preparing transfectant mosaic xenografts composed of a range of proportions of NAT-expressing cells.

The clinical diagnosis of neuroblastoma currently involves histological examination of biopsy specimens and diagnostic MIBG scintigraphy (19) . Diagnostic scintigraphy involves the administration of a tracer dose of radioiodinated MIBG. In addition to providing an indication of the suitability of patients for [131I]MIBG therapy, scintigraphy allows the localization of metastatic deposits and (if supplemented by information allowing the estimation of tumor size) also enables dosimetric calculation. The assay detailed in this report could support the data obtained by MIBG scintigraphy by identifying those patients most suitable for MIBG therapy as a front-line treatment.

Assays based on real-time PCR methodology may also facilitate the development of novel therapies designed to alter cellular function at the transcriptional level (20) . For example, several studies have indicated that treatment of neuroblastoma cells with various genotoxic agents can result in increased [131I]MIBG uptake by enhanced expression of NAT (21, 22, 23, 24) . Whereas the molecular mechanisms involved have yet to be elucidated, the assay described here has the potential to allow quantitative analysis of the effects of these agents on NAT expression and enable the design of improved therapeutic schedules. Studies further investigating the effects of combinations of cytotoxic drugs on NAT mRNA expression are currently under way.

We are now examining the capacity of the real-time PCR assay to accurately quantify NATexpression by tumors to allow the estimation of targeted radiation dose to tumor. Should the absolute level of NATgene expression correlate with MIBG uptake, this assay may prove to be superior to the quantitative power of current dosimetric methodology. It is expected that the outcome of these investigations will be an improved outlook for neuroblastoma patients by virtue of rapid initiation of individualized therapy and the design of optimal combination strategies for cure.

Our results indicate that the real-time PCR method affords accurate and reproducible assessment of the level of NAT transcription and that the detection of NAT mRNA expression in cell lines is predictive of MIBG uptake. The molecular assay may also be capable of predicting the extent of MIBG uptake by tumors in situ. Confirmation of this requires a comparison of PCR data with estimates of MIBG accumulation per unit mass. This, in turn, would necessitate concomitant calculation of tumor size (for example, by computed tomography scanning or magnetic resonance imaging). Such information was not available during the present investigation. Therefore our study was limited to a comparison of scintigraphic positivity or negativity of tumors with NAT expression or failure to express NAT in tumor samples. It could be that later, when we are able to quantify MIBG uptake by quantitative positron emission tomography scanning using [124I]MIBG, we will find a closer correlation between level of NAT expression and MIBG positivity in patients.

In conclusion, our results indicate that NAT-specific, real-time PCR evaluation of primary neuroblastoma tumors has significant capability to reflect tumor capacity for the accumulation of MIBG. Whereas this assay will be useful in preclinical investigations of therapeutic agents that may modulate NAT activity, it is not yet expected to influence the administration of treatment. Prospective studies are needed to assess more precisely the prognostic ability of the real-time PCR examination and its utility in the management of patients.


   ACKNOWLEDGMENTS
 
We are grateful to the following United Kingdom Children’s Cancer Study Group centers for providing tumor samples and copies of diagnostic MIBG scans: Bristol; Edinburgh; Glasgow; Leeds; Liverpool; London (Great Ormond Street and the Royal Marsden Hospitals); Newcastle; Sheffield; and Southampton. Likewise we thank Dutch Children’s Oncology Group centers (Amsterdam, Rotterdam, and Nijmegen) and the G. Gaslini Institute for the supply of tissue samples. We thank Prof. A. J. Malcolm for histological assessment of the tumor content of biopsy samples.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by European Community Project Grant BMH4-CT98-3297, the Neuroblastoma Society, the North of England Children’s Cancer Study Group, and the Cancer Research Campaign. Back

2 Present address: Department of Radiology, Duke University Medical Center, Box 3808, Durham, NC 27710. Back

3 To whom requests for reprints should be addressed, at Department of Radiation Oncology, University of Glasgow, Cancer Research UK Beatson Laboratories, Garscube Estate, Glasgow G61 1BD, United Kingdom. Phone: 44-41-330-4128; Fax: 44-41-330-4127; E-mail: r.mairs{at}beatson.gla.ac.uk Back

4 The abbreviations used are: NAT, noradrenaline transporter; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MIBG, meta-iodobenzylguanidine; hNAT, human NAT; RT-PCR, reverse transcription-PCR; CT, threshold cycle. Back

Received 11/11/02; revised 2/24/03; accepted 2/24/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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