Real-time quantitative reverse transcription-PCR assay for renal cell carcinoma-associated antigen G250.
OBJECTIVE
Gene amplification/expression of G250 is a major event in human renal tumorigenesis. G250-based therapeutic agents and G250-specific gene therapy are under development. These new perspectives call for a sensitive and accurate method to screen G250 alterations in renal cell cancer (RCC) patients and investigate the relationship between G250 mRNA expression and RCC.


METHODS
We developed a quantitative RT-PCR assay for the measurement of G250 mRNA expression using a real-time procedure based on the use of fluorogenic probes and the ABI PRISM 7700 Sequence Detector System. The method has been applied to the measurement of quantitative mRNA level of G250 in 31 cases RCC and 6 normal renal tissues.


RESULTS
The dynamic range was 10(3)-10(8). The relationship between Ct and log starting concentration was linear (r=0.99). G250 expression was present in all RCCs with G250 amplification but was absent in normal ones. G250 mRNA expression ranged from 2.9 x 10(3) to 6.5 x 10(7) copy/microg RNA, with a mean value of 3.5 x 10(6) copy/microg RNA. The expression of G250 revealed an inverse correlation to tumor grade. G250 mRNA level did not correlate with the cell types and clinical stages (P>0.05).


CONCLUSIONS
G250 has the potential to be used as a marker of diagnosis and increasing proliferation in RCC. This new simple, rapid, semi-automated assay was a major alternative to competitive PCR and Northern blot analysis for gene alteration analysis in human tumors and might be a powerful tool for large randomized, prospective cooperative group trials and supporting future G250-based biological and gene therapy approaches.
Chuanzhong
Ye
Y
Department of Urology, Zhong shan Hospital, Medical Center of Fudan University, 180 Feng lin Road, Shanghai 200032, People's Republic of China. chuanzhong@mycity.com.cn
Ming
Guan
G
Fanglin
Zhang
Z
Haijiao
Chen
C
Zhen
Lin
L
Shiping
Chen
C
YongKang
Zhang
Z
D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D000970:Antineoplastic Agents / Q000032:analysis*; D002292:Carcinoma, Renal Cell / Q000276:immunology* / Q000473:pathology; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis; D006801:Humans; D007680:Kidney Neoplasms / Q000276:immunology* / Q000473:pathology; D009367:Neoplasm Staging / Q000379:methods; D011786:Quality Control; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D017931:DNA Primers; D018076:DNA, Complementary; C106533:G250 monoclonal antibody; D012333:RNA, Messenger