- Email: [email protected]
Evaluation of standard tyrosinase RT-PCR in melanoma patients by the use of the LightCycler™ system
Clinica Chimica Acta 306 Ž2001. 133–138 www.elsevier.comrlocaterclinchim
Short communication
Evaluation of standard tyrosinase RT-PCR in melanoma patients by the use of the LightCyclere system Konstantin Stoitchkov a,) , Sabine Letellier b,c , Jean-Pierre Garnier b,c , Miglena Toneva d , Elissaveta Naumova d , Ekaterina Peytcheva a , Nikolai Tzankov e, Bernard Bousquet b,c , Patrice Morel f , Thierry Le Bricon b b
f
a Department of Dermatology, National Cancer Center, PloÕdiÕsko pole Str. No. 6, Sofia 1756, Bulgaria Laboratory of Biochemistry A, Saint-Louis Hospital, AP-HP, 1 AÕenue Claude Vellefaux, 75010 Paris, France c Faculty of Pharmaceutical and Biological Sciences, Paris V UniÕersity, AÕenue de l’obserÕatoire, France d Laboratory of Clinical Immunology, Georgi Sofiiski Str. No. 1, Sofia 1000, Bulgaria e Department of Dermatology, Medical UniÕersity, Georgi Sofiiski Str. No. 1, Sofia 1000, Bulgaria Department of Dermatology B, Saint-Louis Hospital, AP-HP, 1 AÕenue Claude Vellefaux, 75010 Paris, France
Received 17 July 2000; received in revised form 19 December 2000; accepted 30 December 2000
Abstract Background: Haematogenous spread influences outcome in melanoma patients. The clinical relevance of detecting circulating melanoma cells in peripheral blood by tyrosinase mRNA RT-PCR is, however, questioned as rates of positivity considerably vary between studies. Standard tyrosinase-nested RT-PCR was here compared with a real-time PCR technique. Methods: Forty-three blood samples from 20 stage III–IV melanoma patients were analyzed. Mononuclear cells were isolated using a Ficoll Hypaque gradient technique. Total RNA extracted by the acid guanidinum thiocyanate-phenol-chloroform method was reverse transcribed using random hexamers or specific primers. Standard tyrosinase-nested PCR was performed on Touchdowne machine ŽHybaid. and real-time PCR on a LightCyclere instrument ŽRoche.. Results: Only two samples from stage IV patients Žone from random hexamers, one from antisense primers. were found tyrosinase positive with a 100% agreement between the two PCR techniques. A 10-fold dilution of the first-round products improved the PCR kinetic and the final amount of amplified product of positive samples, but not the rate of positivity. Conclusions: Efficiency of the PCR reaction can be monitored in an online fashion by the LightCyclere instrument allowing technical improvements. However , tyrosinase mRNA RT-PCR cannot be yet considered as a useful technique in the monitoring of melanoma patients. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Melanoma; Tyrosinase; RT-PCR; LightCyclere system
1. Introduction )
Corresponding author. Tel.: q359-272-9916; fax: q359-2720651. E-mail addresses: [email protected], [email protected] ŽK. Stoitchkov..
Melanoma is the most aggressive skin cancer with a doubling incidence every 10 years for the Caucasian population worldwide w1x. Haematogenous
0009-8981r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 Ž 0 1 . 0 0 4 0 0 - 4
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K. StoitchkoÕ et al.r Clinica Chimica Acta 306 (2001) 133–138
spread strongly influences patient outcome, but there is to date no reliable laboratory test for early detection and monitoring of disease progression. RT-PCR technology has made the search for circulating melanoma cells in peripheral blood of melanoma patients possible w2x. Tyrosinase, an enzyme involved in melanogenesis, appears the most reliable mRNA target w3–5x, since it is a specific marker of melanocytic differentiation, expressed both in primary and metastatic melanoma. In vitro Žspiking. studies have demonstrated the sensitivity Ž1 melanoma cell in 10 ml of blood. of currently applied tyrosinase RT-PCR protocols w4x. The clinical relevance of in vivo tyrosinase mRNA analysis in melanoma patients is, however, strongly questioned Table 1 Study design
as rates of positivity even in stage IV patients Žclassification according to the American Joint Committee on Cancer Staging-AJCC of melanoma. w6x vary between 15% and 100% w4,8,9x. We w8x and others w7,10x reported a low rate of positivity in stage III–IV melanoma patients using a well-standardized and sensitive Ž10y6 mgrmg control RNA. nested PCR protocol. This low rate might be attributed to the heterogeneous panel of patients within the same stage, absence of circulating melanoma cells at the time of sampling, inappropriate blood processing andror unoptimized RT-PCR protocols. In the present study, standard tyrosinase-nested PCR w8x was compared with the LightCyclere realtime PCR in stage III–IV melanoma patients. The
K. StoitchkoÕ et al.r Clinica Chimica Acta 306 (2001) 133–138
LightCyclere technology combines rapid thermocycling with online fluorescence detection of the PCR amplicon providing critical information on the overall quality of the PCR reaction.
2. Material and methods 2.1. Patients Twenty melanoma patients Ž9 Mr11 F, age: 29– 73 years. entered the study after histological diagnosis and staging according to the classification of the AJCC Žstage III: n s 14, stage IV: n s 6.. All patients were diagnosed, treated, followed up and sampled at the Department of Dermatology at the National Cancer Center in Sofia, Bulgaria. They were sampled at the moment of diagnosis, after treatments Žsurgical or chemotherapyrimmunotherapy. and during follow up, so a total of 43 blood samples Žstage III: n s 28, stage IV: n s 15. were analyzed by tyrosinase RT-PCR ŽTable 1.. Ten healthy subjects Ž5 Mr5 F, age: 25–71 years. and six nonmelanoma skin cancer patients Ž3 Mr3 F, age: 30–62 years. were also included as controls.
135
200 pmol of outer antisense primers ŽGenset, Paris, France. for 1 h at 428C ŽTable 1.. Final volumes were 50 ml. PCR analyses of tyrosine transcripts were performed using nested RT-PCR with primers described by Smith et al. w2x Žfinal PCR product: 207 bp.. A standard nested RT-PCR was performed using a Touchdowne machine ŽHybaid, Middlesex, UK. in the conditions previously described by us w8x. Realtime PCR was realized on a LightCyclere instrument ŽRoche, Mannheim, Germany. with primers obtained from Metabion ŽMartinsried, Germany.. In this system, the PCR reaction is monitored using two hybridization probes Ž3F1, 5LC. labeled with fluorescein Ždonor dye. or LC red 640: Žacceptor dye. allowing a fluorescence resonance energy transfer after hybridization to the target sequences on the amplified DNA fragment. Intensity of the light emit-
2.2. Blood sampling and processing All procedures have been previously described in detail w8x. Briefly, 10 ml of blood was drawn by venous puncture in tubes with EDTA as an anticoagulant. The first 3 ml was discarded to avoid contamination by skin melanocytes. Mononuclear cells were isolated using a Ficoll Hypaque gradient technique followed by total cellular RNA extraction using the original acid guanidinium thiocyanate-phenol-chloroform method w11x. Concentration, purity and integrity of total RNA were determined by UV spectrophotometry, 2% agarose gel electrophoresis and RT-PCR with primers for b2-microglobulin producing a fragment of 620 bp w8x. 2.3. RT-PCR One microgram total RNA was reverse transcribed using random hexamers ŽPharmacia. for 1 h at 378C as described previously w8x and also using
Fig. 1. ŽA. Standard ŽTouchdown HYBAIDe. PCR for b2-microglobulin, MWs molecular weight marker, 1s cDNA of tyrosinase positive patient, 2 s cDNA of tyrosinase negative patient, TŽq. s positive control, TŽy. s negative control. ŽB. Standard ŽTouchdown HYBAIDe. PCR for tyrosinase, MWs molecular weight marker, 1s cDNA of tyrosinase positive patient, 2 s cDNA of tyrosinase negative patient, TŽq. s positive control, TŽy. s negative control.
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K. StoitchkoÕ et al.r Clinica Chimica Acta 306 (2001) 133–138
ted by LC red 640 is proportional to DNA formation and is measured at 640 nm. Briefly, two microliters of cDNA was amplified in a final volume of 20 ml using the 10X Light cycler DNA master hybridization probes kit containing ready-to-use reaction mix for PCR Žtaq polymerase, buffer, deoxynucleoside triphosphate and 10 mmolrl MgCl 2 . ŽRoche.. After dilution to 1X and adjustment of MgCl 2 concentration to 3 mmolrl, the final PCR mixture contained 12.5 pmol of primers Žouter or inner. and 4 pmol of each probe. PCR conditions were 958C for 2 min, 30 cycles at 958C for 45 s, 608C for 45 s and 728C for 50 s, then 728C for 5 min. For nested PCR, 30 additional cycles were added using Ar2 ml of the first-round product or Br2 ml of a 10-fold dilution in the PCR buffer.
3. Results Total mRNA extracted from buffy coats ranged from 2 to 52 mg Žmean value: 16 mg. with a mean 260r280 nm ratio of 1.702. Only two samples needed to be reextracted Ž260r280 nm ratio - 1.6.. All sam-
Fig. 2. LightCyclere PCR kinetics of tyrosinase assay. The first-round product was used for second-round PCR—undiluted Žopen symbols. and after 1r10-fold dilution in PCR buffer Žblack symbols..
ples showed 28 S and 18 S ribosomal bands and were found positive for b2-microglobulin PCR amplification ŽFig. 1A.. None of the healthy subjects and nonmelanoma skin cancer patients tested positive for tyrosinase mRNA by standard and real-time PCR, so that specificity was 100%. All samples from stage III patients were negative for tyrosinase mRNA by standard and real-time PCR. Two samples from stage IV patients ŽFig. 1B. were found tyrosinase positive by standard PCR and by the LightCyclere system. One positive sample was obtained from cDNA reverse transcribed with random hexamers and the other one using antisense primers with a 100% agreement between the two PCR techniques. A 10-fold dilution of the first-round products significantly improved the PCR kinetic of positive samples and the final amount of amplified product ŽFig. 2.. It had, however, no effect on all negative samples, which remained negative.
4. Discussion In the present study, we used the LightCyclere technology to evaluate our standard nested PCR technique, sensitive but associated with a low rate of positivity in stage III–IV melanoma patients w8x. In the present study, each step from blood processing to final visualization of PCR amplicon was carefully selected according to recent advances reported in the literature. Total mRNA from potentially present melanoma cells is usually obtained from buffy coat after gradient centrifugation or directly after whole blood processing. We selected a Ficoll-Hypaque gradient technique for blood processing according to our previous experience w8x and others w12,13x. Gradient centrifugation is convenient, easy to run and significantly reduces the volumes of reagents compared to whole blood processing. Importantly, the sensitivity of RTPCR following a gradient technique was recently found to be superior to that of whole blood in an in vitro spiking experiment w14x. We here also confirm that the original method of Chomczynski and Sacchi w11x using guanidinium isothiocyanate-phenol-
K. StoitchkoÕ et al.r Clinica Chimica Acta 306 (2001) 133–138
chloroform-based RNA extraction, while laborious and time consuming, provides excellent results from buffy coats of melanoma patients in terms of RNA quality Ž- 5% reextraction, 16 mg total RNA.. All 43 RNAs were successfully reverse transcribed and amplified for human b2-microglobulin. It has been, however, recently reported that low rates of melanoma markers mRNA positivity might not be related to differences in mRNA quality w15x. Reverse transcription protocols are relatively similar among studies, often using the Moloney Murine Leukemia Virus ŽMMLV. reverse transcriptase Žpreferred for its RNAse inhibiting activity. and random oligonucleotides sequences rather than specific primers to generate cDNA as it allows RT-PCR on different targets. Our results suggest that with both types of primers, positive signals can be detected or missed, so it would be more appropriate to do both. The only method available to evaluate RT quality is running a PCR for ubiquitous housekeeping genes Žsuch as b2-microglobulin.. The major problem with those genes is that their expression might be detected in poor quality cDNA andror low efficiency RT. Monitoring of PCR kinetics of ubiquitous genes by the LightCyclere system could provide more valuable information on cDNA quality. Tyrosinase-nested PCR is usually performed with two successive amplifications using primers developed by Smith et al. w2x. Protocols, however, differ in reagents, PCR conditions and methods for visualization of PCR amplicons. We here reproduced most of these interlaboratory variations without any impact on PCR positivity. A 10-fold dilution of the firstround PCR product did not change the overall result, despite significant improvements in PCR kinetics and final amount of amplified products in positive samples. Nested PCR is often performed after dilution of the first-round products for a maximum efficacy during the second-round amplification. Using real-time PCR, we here verified the validity of this approach Ža 1:10 dilution. for the detection of tyrosinase mRNA in peripheral blood of melanoma patients. The detection of amplified products by hybridization probes in the LightCyclere system was also significantly improved over ethidium bromide staining, but not the rate of positivity. Post PCR Southern blotting w16x or electrochemiluminiscence Žbiotinylated post PCR cDNA samples are hy-
137
bridized to a chelate-labeled oligonucleotide probe. w17x has been proposed to increase detection sensitivity. Here, the two positive samples were found in patients with evolutive disease, who had more than one distant metastatic site and who died within 2 months following the test positivity.
5. Conclusion The LightCyclere appears as a suitable system for an easy-to-run RT-PCR technique for tyrosinase mRNA amplification in melanoma patients. The efficiency and overall quality of the PCR reactions can be monitored in an online fashion allowing technical improvements and further quantification of amplified PCR products. The standard or real-time tyrosinase RT-PCR assay, however, cannot yet be considered as a useful marker in the monitoring of melanoma patients.
Acknowledgements We would like specially to thank Nicole Max and Ulrich Keilholz from the Department of Hematology, Oncology and Transfusion at Benjamin Franklin University Hospital, Free University of Berlin, Germany, for kindly providing us the opportunity to use the LightCyclere system.
References w1x Caron M, Jorgensen G, Rigel D, Friedman R. In: Blach CM, Houghton AN, Miltron GM, editors. The Worldwide Incidence of Malignant Melanoma. Philadelphia: JB Lippincott, 1992:27–45. w2x Smith B, Selby P, Southgate J, Pittman K, Bradley C, Blair GE, et al. Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction. Lancet 1991;338:1227–9. w3x Hoon DSB, Wang Y, Dale PS, et al. Detection of occult melanoma cells in blood with a multiple-marker polymerase chain reaction assay. J Clin Oncol 1995;13:2109–16. w4x Keilhoiz U. New prognostic factors in melanoma: mRNA tumor markers. Eur J Cancer 1998;l34ŽSuppl. 3.:S37–41. w5x Muhlbauer M, Langenbach N, Stolz W, et al. Detection of ¨ melanoma cells in the blood of melanoma patients by melanoma-inhibitory activity ŽMIA. reverse-transcription PCR. Clin Cancer Res 1999;5:1099–105.
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w6x Beahrs OH, Myers MH. Manual for Staging of Cancer. 4th edn. Philadelphia, PA: JB Lippincott, 1995:1555–60. w7x Keilholz U, Willhauck M, Scheibenbogen C, De Vries TJ, Burchill S. Polymerase chain reaction detection of circulating tumor cells. Melanoma Res 1997;7ŽSuppl. 2.:s133–41. w8x Le Bricon T, Stoitchkov K, Letellier S, et al. Simultaneous analysis of tyrosinase mRNA and markers of tyrosinase activity in the blood of patients with metastatic melanoma. Clin Chim Acta 1999;282:101–13. w9x Aubin F, Chtourou M, Teyssier JR, et al. The detection of tyrosinase mRNA in the peripheral blood of stage I melanoma patients is not of clinical relevance in predicting metastasis risk and survival. Melanoma Res 2000;10:113–8. w10x Reinhold U, Ludke-Handjery H-C, Schnautz S, Kreysel HW, ¨ Abken H. The analysis of tyrosinase-specific mRNA in blood samples of melanoma patients by RT-PCR is not a useful test for metastatic tumor progression. J Invest Dermatol 1997; 108:166–9. w11x Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Annal Biochem 1987;162:156–9. w12x Jung F, Buzaid A, Ross M, et al. Evaluation of tyrosinase mRNA as a tumor marker in the blood of melanoma patients. J Clin Oncol 1997;15:2826–31. w13x Glaser R, Rass K, Seiter S, Hauschild A, Christophers E,
w14x
w15x
w16x
w17x
Tilgen W. Detection of circulating melanoma cells by specific amplification of tyrosinase complementary DNA is not a reliable tumor marker in melanoma patients: a clinical two center study. J Clin Oncol 1997;15:2818–25. De Vries TJ, Fourkour A, Punt CJA, Ruiter DJ, Van Muijen GNP. Analysis of melanoma cells in peripheral blood by reverse transcription-polymerase chain reaction for tyrosinase and MART-1 after mononuclear cell collection with cell preparation tubes: a comparison with the whole blood guanidinium isothiocyanate RNA isolation method. Melanoma Res 2000;10:119–26. De Vries TJ, Fourkour A, Punt CJA, et al. Reproducibility of detection of tyrosinase and MART-1 transcripts in the peripheral blood of melanoma patients: quality control study using real-time quantitative RT-PCR. Br J Cancer 1999; 80:883–91. Brossart P, Schmier JW, Kruger S, et al. A polymerase chain reaction-based semiquantitative assessment of malignant melanoma cells in peripheral blood. Cancer Res 1995; 55:4065–8. O’Connell C, Juhasz A, Kuo C, Reeder D, Hoon D. Detection of tyrosinase mRNA in melanoma by reverse transcription PCR and electrochemiluminescence. Clin Chem 1998;44:1161–9.
Short communication
Evaluation of standard tyrosinase RT-PCR in melanoma patients by the use of the LightCyclere system Konstantin Stoitchkov a,) , Sabine Letellier b,c , Jean-Pierre Garnier b,c , Miglena Toneva d , Elissaveta Naumova d , Ekaterina Peytcheva a , Nikolai Tzankov e, Bernard Bousquet b,c , Patrice Morel f , Thierry Le Bricon b b
f
a Department of Dermatology, National Cancer Center, PloÕdiÕsko pole Str. No. 6, Sofia 1756, Bulgaria Laboratory of Biochemistry A, Saint-Louis Hospital, AP-HP, 1 AÕenue Claude Vellefaux, 75010 Paris, France c Faculty of Pharmaceutical and Biological Sciences, Paris V UniÕersity, AÕenue de l’obserÕatoire, France d Laboratory of Clinical Immunology, Georgi Sofiiski Str. No. 1, Sofia 1000, Bulgaria e Department of Dermatology, Medical UniÕersity, Georgi Sofiiski Str. No. 1, Sofia 1000, Bulgaria Department of Dermatology B, Saint-Louis Hospital, AP-HP, 1 AÕenue Claude Vellefaux, 75010 Paris, France
Received 17 July 2000; received in revised form 19 December 2000; accepted 30 December 2000
Abstract Background: Haematogenous spread influences outcome in melanoma patients. The clinical relevance of detecting circulating melanoma cells in peripheral blood by tyrosinase mRNA RT-PCR is, however, questioned as rates of positivity considerably vary between studies. Standard tyrosinase-nested RT-PCR was here compared with a real-time PCR technique. Methods: Forty-three blood samples from 20 stage III–IV melanoma patients were analyzed. Mononuclear cells were isolated using a Ficoll Hypaque gradient technique. Total RNA extracted by the acid guanidinum thiocyanate-phenol-chloroform method was reverse transcribed using random hexamers or specific primers. Standard tyrosinase-nested PCR was performed on Touchdowne machine ŽHybaid. and real-time PCR on a LightCyclere instrument ŽRoche.. Results: Only two samples from stage IV patients Žone from random hexamers, one from antisense primers. were found tyrosinase positive with a 100% agreement between the two PCR techniques. A 10-fold dilution of the first-round products improved the PCR kinetic and the final amount of amplified product of positive samples, but not the rate of positivity. Conclusions: Efficiency of the PCR reaction can be monitored in an online fashion by the LightCyclere instrument allowing technical improvements. However , tyrosinase mRNA RT-PCR cannot be yet considered as a useful technique in the monitoring of melanoma patients. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Melanoma; Tyrosinase; RT-PCR; LightCyclere system
1. Introduction )
Corresponding author. Tel.: q359-272-9916; fax: q359-2720651. E-mail addresses: [email protected], [email protected] ŽK. Stoitchkov..
Melanoma is the most aggressive skin cancer with a doubling incidence every 10 years for the Caucasian population worldwide w1x. Haematogenous
0009-8981r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 Ž 0 1 . 0 0 4 0 0 - 4
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K. StoitchkoÕ et al.r Clinica Chimica Acta 306 (2001) 133–138
spread strongly influences patient outcome, but there is to date no reliable laboratory test for early detection and monitoring of disease progression. RT-PCR technology has made the search for circulating melanoma cells in peripheral blood of melanoma patients possible w2x. Tyrosinase, an enzyme involved in melanogenesis, appears the most reliable mRNA target w3–5x, since it is a specific marker of melanocytic differentiation, expressed both in primary and metastatic melanoma. In vitro Žspiking. studies have demonstrated the sensitivity Ž1 melanoma cell in 10 ml of blood. of currently applied tyrosinase RT-PCR protocols w4x. The clinical relevance of in vivo tyrosinase mRNA analysis in melanoma patients is, however, strongly questioned Table 1 Study design
as rates of positivity even in stage IV patients Žclassification according to the American Joint Committee on Cancer Staging-AJCC of melanoma. w6x vary between 15% and 100% w4,8,9x. We w8x and others w7,10x reported a low rate of positivity in stage III–IV melanoma patients using a well-standardized and sensitive Ž10y6 mgrmg control RNA. nested PCR protocol. This low rate might be attributed to the heterogeneous panel of patients within the same stage, absence of circulating melanoma cells at the time of sampling, inappropriate blood processing andror unoptimized RT-PCR protocols. In the present study, standard tyrosinase-nested PCR w8x was compared with the LightCyclere realtime PCR in stage III–IV melanoma patients. The
K. StoitchkoÕ et al.r Clinica Chimica Acta 306 (2001) 133–138
LightCyclere technology combines rapid thermocycling with online fluorescence detection of the PCR amplicon providing critical information on the overall quality of the PCR reaction.
2. Material and methods 2.1. Patients Twenty melanoma patients Ž9 Mr11 F, age: 29– 73 years. entered the study after histological diagnosis and staging according to the classification of the AJCC Žstage III: n s 14, stage IV: n s 6.. All patients were diagnosed, treated, followed up and sampled at the Department of Dermatology at the National Cancer Center in Sofia, Bulgaria. They were sampled at the moment of diagnosis, after treatments Žsurgical or chemotherapyrimmunotherapy. and during follow up, so a total of 43 blood samples Žstage III: n s 28, stage IV: n s 15. were analyzed by tyrosinase RT-PCR ŽTable 1.. Ten healthy subjects Ž5 Mr5 F, age: 25–71 years. and six nonmelanoma skin cancer patients Ž3 Mr3 F, age: 30–62 years. were also included as controls.
135
200 pmol of outer antisense primers ŽGenset, Paris, France. for 1 h at 428C ŽTable 1.. Final volumes were 50 ml. PCR analyses of tyrosine transcripts were performed using nested RT-PCR with primers described by Smith et al. w2x Žfinal PCR product: 207 bp.. A standard nested RT-PCR was performed using a Touchdowne machine ŽHybaid, Middlesex, UK. in the conditions previously described by us w8x. Realtime PCR was realized on a LightCyclere instrument ŽRoche, Mannheim, Germany. with primers obtained from Metabion ŽMartinsried, Germany.. In this system, the PCR reaction is monitored using two hybridization probes Ž3F1, 5LC. labeled with fluorescein Ždonor dye. or LC red 640: Žacceptor dye. allowing a fluorescence resonance energy transfer after hybridization to the target sequences on the amplified DNA fragment. Intensity of the light emit-
2.2. Blood sampling and processing All procedures have been previously described in detail w8x. Briefly, 10 ml of blood was drawn by venous puncture in tubes with EDTA as an anticoagulant. The first 3 ml was discarded to avoid contamination by skin melanocytes. Mononuclear cells were isolated using a Ficoll Hypaque gradient technique followed by total cellular RNA extraction using the original acid guanidinium thiocyanate-phenol-chloroform method w11x. Concentration, purity and integrity of total RNA were determined by UV spectrophotometry, 2% agarose gel electrophoresis and RT-PCR with primers for b2-microglobulin producing a fragment of 620 bp w8x. 2.3. RT-PCR One microgram total RNA was reverse transcribed using random hexamers ŽPharmacia. for 1 h at 378C as described previously w8x and also using
Fig. 1. ŽA. Standard ŽTouchdown HYBAIDe. PCR for b2-microglobulin, MWs molecular weight marker, 1s cDNA of tyrosinase positive patient, 2 s cDNA of tyrosinase negative patient, TŽq. s positive control, TŽy. s negative control. ŽB. Standard ŽTouchdown HYBAIDe. PCR for tyrosinase, MWs molecular weight marker, 1s cDNA of tyrosinase positive patient, 2 s cDNA of tyrosinase negative patient, TŽq. s positive control, TŽy. s negative control.
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K. StoitchkoÕ et al.r Clinica Chimica Acta 306 (2001) 133–138
ted by LC red 640 is proportional to DNA formation and is measured at 640 nm. Briefly, two microliters of cDNA was amplified in a final volume of 20 ml using the 10X Light cycler DNA master hybridization probes kit containing ready-to-use reaction mix for PCR Žtaq polymerase, buffer, deoxynucleoside triphosphate and 10 mmolrl MgCl 2 . ŽRoche.. After dilution to 1X and adjustment of MgCl 2 concentration to 3 mmolrl, the final PCR mixture contained 12.5 pmol of primers Žouter or inner. and 4 pmol of each probe. PCR conditions were 958C for 2 min, 30 cycles at 958C for 45 s, 608C for 45 s and 728C for 50 s, then 728C for 5 min. For nested PCR, 30 additional cycles were added using Ar2 ml of the first-round product or Br2 ml of a 10-fold dilution in the PCR buffer.
3. Results Total mRNA extracted from buffy coats ranged from 2 to 52 mg Žmean value: 16 mg. with a mean 260r280 nm ratio of 1.702. Only two samples needed to be reextracted Ž260r280 nm ratio - 1.6.. All sam-
Fig. 2. LightCyclere PCR kinetics of tyrosinase assay. The first-round product was used for second-round PCR—undiluted Žopen symbols. and after 1r10-fold dilution in PCR buffer Žblack symbols..
ples showed 28 S and 18 S ribosomal bands and were found positive for b2-microglobulin PCR amplification ŽFig. 1A.. None of the healthy subjects and nonmelanoma skin cancer patients tested positive for tyrosinase mRNA by standard and real-time PCR, so that specificity was 100%. All samples from stage III patients were negative for tyrosinase mRNA by standard and real-time PCR. Two samples from stage IV patients ŽFig. 1B. were found tyrosinase positive by standard PCR and by the LightCyclere system. One positive sample was obtained from cDNA reverse transcribed with random hexamers and the other one using antisense primers with a 100% agreement between the two PCR techniques. A 10-fold dilution of the first-round products significantly improved the PCR kinetic of positive samples and the final amount of amplified product ŽFig. 2.. It had, however, no effect on all negative samples, which remained negative.
4. Discussion In the present study, we used the LightCyclere technology to evaluate our standard nested PCR technique, sensitive but associated with a low rate of positivity in stage III–IV melanoma patients w8x. In the present study, each step from blood processing to final visualization of PCR amplicon was carefully selected according to recent advances reported in the literature. Total mRNA from potentially present melanoma cells is usually obtained from buffy coat after gradient centrifugation or directly after whole blood processing. We selected a Ficoll-Hypaque gradient technique for blood processing according to our previous experience w8x and others w12,13x. Gradient centrifugation is convenient, easy to run and significantly reduces the volumes of reagents compared to whole blood processing. Importantly, the sensitivity of RTPCR following a gradient technique was recently found to be superior to that of whole blood in an in vitro spiking experiment w14x. We here also confirm that the original method of Chomczynski and Sacchi w11x using guanidinium isothiocyanate-phenol-
K. StoitchkoÕ et al.r Clinica Chimica Acta 306 (2001) 133–138
chloroform-based RNA extraction, while laborious and time consuming, provides excellent results from buffy coats of melanoma patients in terms of RNA quality Ž- 5% reextraction, 16 mg total RNA.. All 43 RNAs were successfully reverse transcribed and amplified for human b2-microglobulin. It has been, however, recently reported that low rates of melanoma markers mRNA positivity might not be related to differences in mRNA quality w15x. Reverse transcription protocols are relatively similar among studies, often using the Moloney Murine Leukemia Virus ŽMMLV. reverse transcriptase Žpreferred for its RNAse inhibiting activity. and random oligonucleotides sequences rather than specific primers to generate cDNA as it allows RT-PCR on different targets. Our results suggest that with both types of primers, positive signals can be detected or missed, so it would be more appropriate to do both. The only method available to evaluate RT quality is running a PCR for ubiquitous housekeeping genes Žsuch as b2-microglobulin.. The major problem with those genes is that their expression might be detected in poor quality cDNA andror low efficiency RT. Monitoring of PCR kinetics of ubiquitous genes by the LightCyclere system could provide more valuable information on cDNA quality. Tyrosinase-nested PCR is usually performed with two successive amplifications using primers developed by Smith et al. w2x. Protocols, however, differ in reagents, PCR conditions and methods for visualization of PCR amplicons. We here reproduced most of these interlaboratory variations without any impact on PCR positivity. A 10-fold dilution of the firstround PCR product did not change the overall result, despite significant improvements in PCR kinetics and final amount of amplified products in positive samples. Nested PCR is often performed after dilution of the first-round products for a maximum efficacy during the second-round amplification. Using real-time PCR, we here verified the validity of this approach Ža 1:10 dilution. for the detection of tyrosinase mRNA in peripheral blood of melanoma patients. The detection of amplified products by hybridization probes in the LightCyclere system was also significantly improved over ethidium bromide staining, but not the rate of positivity. Post PCR Southern blotting w16x or electrochemiluminiscence Žbiotinylated post PCR cDNA samples are hy-
137
bridized to a chelate-labeled oligonucleotide probe. w17x has been proposed to increase detection sensitivity. Here, the two positive samples were found in patients with evolutive disease, who had more than one distant metastatic site and who died within 2 months following the test positivity.
5. Conclusion The LightCyclere appears as a suitable system for an easy-to-run RT-PCR technique for tyrosinase mRNA amplification in melanoma patients. The efficiency and overall quality of the PCR reactions can be monitored in an online fashion allowing technical improvements and further quantification of amplified PCR products. The standard or real-time tyrosinase RT-PCR assay, however, cannot yet be considered as a useful marker in the monitoring of melanoma patients.
Acknowledgements We would like specially to thank Nicole Max and Ulrich Keilholz from the Department of Hematology, Oncology and Transfusion at Benjamin Franklin University Hospital, Free University of Berlin, Germany, for kindly providing us the opportunity to use the LightCyclere system.
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